Article pubs.acs.org/ac
Quantitative Detection of Potassium Ions and Adenosine Triphosphate via a Nanochannel-Based Electrochemical Platform Coupled with G‑Quadruplex Aptamers Jiachao Yu, Linqun Zhang, Xuan Xu, and Songqin Liu* Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Suzhou Research Institute of Southeast University, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, P. R. China S Supporting Information *
ABSTRACT: The development of synthetic nanopores and nanochannels that mimick ion channels in living organisms for biosensing applications has been, and still remains, a great challenge. Although the biological applications of nanopores and nanochannels have achieved considerable development as a result of nanotechnology advancements, there are few reports of a facile way to realize those applications. Herein, a nanochannelbased electrochemical platform was developed for the quantitative detection of biorelated small molecules such as potassium ions (K+) and adenosine triphosphate (ATP) in a facile way. For this purpose, K+ or ATP G-quadruplex aptamers were covalently assembled onto the inner wall of porous anodic alumina (PAA) nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Conformational switching of the aptamers confined in the nanochannels occurs in the presence of the target molecules, resulting in increased steric hindrance in the nanochannels. Changes in steric hindrance in the nanochannels were monitored by the anodic current of indicator molecules transported through the nanochannels. As a result, quantitative detection of K+ and ATP was realized with a concentration ranging from 0.005 to 1.0 mM for K+ and 0.05 to 10.0 mM for ATP. The proposed platform displayed significant selectivity, good reproducibility, and universality. Moreover, this platform showed its potential for use in the detection of other aptamer-based analytes, which could promote its development for use in biological detection and clinical diagnosis.
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DNA sequences and detecting proteins and other analytes.13−15 For single nanochannel-based nanodevices, the typical strategy involves the measurement of electrical conductance between two sides of the membrane, which changes based on the presence of molecules in the nanochannel.16,17 As for nanochannel array-based nanodevices, the nanochannel arrays are first modified with biorecognition molecules and are then used as a coating on the surface of conventional electrotransducers.18 By monitoring the variations in the electrochemical signals of the indicator molecules transported through the nanochannels, the presence of target molecules in the nanochannels could be determined.19 The fundamental mechanism of these nanochannel-based applications is the transport of indicator molecules or ions inside the nanochannels. Such transport is generally affected by two factors: steric hindrance and electrostatic repulsion.19 Steric hindrance is induced by the space occupied by the biomolecules inside the nanochannels.17 Because the size of the biomolecules
ecently, the development of synthetic nanopores and nanochannels that mimick the ion channels in living organisms and biosensing applications has attracted considerable attention.1−4 In living systems, the selective transportation of ions in protein-based ion channels is the source of electrical signals in nerves and muscles. This natural behavior inspired the application of nanochannels to biosensor development. The pioneer works in this field were realized by inserting a natural protein ion channel, such as α-hemolysin, into lipid bilayers to form a single biological nanochannel.5 The ionic current through such nanochannels could be altered when a target molecule appeared in the specific region of the nanochannel.6 Following this work, a number of innovative nanochannel-based biosensing systems have been developed, with special focus on DNA sequencing.7,8 Very recently, artificial single nanochannels and nanochannel arrays, which were built and embedded in mechanically and chemically robust synthetic membranes, have been prepared by utilizing various materials and technologies.9−12 The shape, size, and length of these nanochannels in membranes could be wellcontrolled during fabrication, demonstrating their remarkable promise for use in nanodevices for biosensing, e.g., monitoring © 2014 American Chemical Society
Received: July 24, 2014 Accepted: October 10, 2014 Published: October 10, 2014 10741
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Scheme 1. Functionalization of PAA Nanochannels with Aptamer and Flux of the Redox Indicator (Hydroquinone) in the Nanochannels before and after Conformational Switching of the Aptamer
bonding interactions, which are promoted by the presence of their targets.34−37 Jiang et al. reported a K+-responsive PET single nanochannel on the basis of G-quadruplex DNA conformational switching.38 In their work, G-quadruplex DNA was immobilized onto a synthetic nanopore, and it underwent a potassium-responsive conformational change that induced a change in the effective pore size. The responsiveness could be regulated by the stability of the G-quadruplex structure through adjusting the potassium concentration, which was monitored by the transmembrane ion current across the PET film. Inspired by potassium-responsive G-quadruplex DNA conformational switching in a nanopore, a nanochannel-based electrochemical platform was developed for the quantitative detection of K+ and adenosine triphosphate (ATP) in a facile way. As shown in Scheme 1, the PAA nanochannels were silanized and then modified with single-stranded K+ or ATP Gquadruplex aptamers through a Schiff reaction. When the target molecules were introduced into the nanochannels, the aptamers fold into G-quadruplex structures. Such conformational switching results in a significant increase in the steric hindrance in the nanochannels, which leads to a decrease in the flux of indicator molecules (hydroquinone, HQ) in the nanochannels. Because the flux of indicator molecules was monitored by their anodic current at the working electrode, the target molecules could be detected in a simple and rapid way.
is non-negligible compared with the pore diameter of the nanochannels, the free transport area for the indicator molecules or ions decreased. Electrostatic repulsion, however, was induced not only by the charge of the biomolecules20 but also the surface charge on the inner wall of the nanochannels.21 These charges generated an electrostatic field inside the nanochannels, which would attract or repel charged molecules and ions, and thus the transport of indicator molecules or ions was affected. On the basis of this principle, unlike stable steric hindrance, electrostatic repulsion can be influenced by the environment, such as the ionic strength and pH of the solution. Porous anodic alumina (PAA) membrane is a popular material for nanochannel-based biosensor design because of its tunable nanopore diameter, well-defined nanochannel array, robust overall structure, ease of surface functionalization, and commercial availability.22,23 For instance, Merkoçi et al.18 reported a novel PAA nanochannel-based biosensor for antigen detection. In their work, the PAA membrane was modified with antibodies, and the conductivity of the membrane toward a redox indicator was tuned by primary and secondary immunoreactions with proteins and gold nanoparticles in the nanochannels. The blockage by gold nanoparticles was enhanced by silver deposition that further decreased the diffusion of the signaling indicator through the nanochannels. The principle of such applications was based on the modulation of indicator molecules transported through the nanochannels, and their applicability depended intimately on the surface characteristics of the nanochannel’s inner walls. K+ and ATP are both essential species in the human body that play fundamental roles in various biological processes.24,25 Abnormal concentrations of both of these species are tightly associated with a series of diseases.26,27 Thus, the detection of K+ and ATP is vitally important for biochemical studies and clinic diagnosis.28,29 Aptamers are artificial DNA or RNA oligonucleotides that can bind to a wide variety of targets with high affinity and selectivity.30−33 In particular, single-stranded DNA aptamers with a guanine (G)-rich sequence can be folded into G-quadruplex structures via intramolecular hydrogen-
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EXPERIMENTAL SECTION Materials. K+ aptamer (5′-CHO-AAA AAA AAA AGG GTT AGG GTT AGG GTT AGG G-3′) and ATP aptamer (5′-CHO-AAA AAA AAA ACC TGG GGG AGT ATT GCG GAG GAA GG-3′) were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Porous anodic alumina (PAA) was purchased from Hefei Puyuan Nanotechnology Co. Ltd. (Anhui, China). (3-Aminopropyl)triethoxysilane (APTES), adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) were received from Sigma-Aldrich (Shanghai, China). 10742
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well as a Pt counter electrode and a saturated calomel electrode (SCE) over the PAA membrane formed a three-electrode electrochemical system. To illustrate the steric hindrance in PAA nanochannels before and after the conformational switching of the aptamer, the electrolyte containing 1 mM neutrally charged HQ was used. Agitation was applied to the electrolyte before the electrochemical measurement in order to form a homogeneous HQ concentration inside and outside the nanochannels. As shown in Scheme 1, the consumption of HQ at the working electrode decreased the concentration of HQ inside the nanochannels, which facilitated the diffusion of HQ molecules in the bulk electrolyte into the nanochannels. Thus, a flux of HQ in the nanochannels formed, and the diffusionlimited steady-state flux, which reflects the steric hindrance in the nanochannels, was indicated by the steady-state anodic current of HQ (Figure S1). It should be noted that the current did not reach a strict steady state in an acceptable amount of time. To balance these two factors, all measurements were stopped at 600 s, and the currents were consistently read at 600 s. CD Spectroscopy Measurements. CD spectra were collected on a Chirascan CD spectrometer (Applied Photophysics Ltd., UK). Wavelength scans were performed between 220 and 320 nm. A quartz cell with a path length of 10 mm was used. The Tris buffer solutions (10 mM, pH 7.4), containing 5 μM aptamer and various concentrations of K+ or ATP, were incubated at room temperature for 1 h before CD spectroscopy measurements.
Hydroquinone (HQ), LiCl, NaCl, KCl, RbCl, and CsCl were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and used as received. Ultrapure water (Thermo Fisher Barnstead, 18.2 MΩ) was used throughout the study. Covalent Linking of the Aptamer to PAA Nanochannels. The functionalization of PAA nanochannels with aptamer is illustrated in Scheme 1. The morphology and size of PAA membranes were characterized by field-emission scanning electron microscopy (SEM, ULTRA PLUS, ZEISS, Germany) at an acceleration voltage of 5 kV. PAA membranes with a thickness of 81 μm, a diameter of 5 mm, a geometry area of 19.6 mm2, and pore diameters of 25 ± 5, 55 ± 15, 90 ± 10, and 130 ± 20 nm were washed by ethanol and ultrapure water to remove any impurities in the nanochannels. After drying in a nitrogen flow at room temperature, the PAA membranes were immersed into 1 mL of an ethanol solution containing 5% APTES and shaken gently for 12 h to generate −NH2 groups on the inner wall of the PAA nanochannels.39 Then, the PAA membranes were washed with ethanol to remove any residual silane in the nanochannels and dried in a nitrogen flow at room temperature. After that, 10 μL of a 100 μM aptamer aqueous solution was dropped onto the surface of the PAA membrane and allowed to react for 24 h.40 It should be noted that the PAA membrane was placed on a frame that was fixed in an air-tight glass bottle with some water at the bottom. The saturated vapor pressure in the bottle prevented the 10 μL aqueous solution from evaporation over 24 h. The remaining amino groups were blocked by immersing the PAA membrane into 1 mL of ultrapure water containing 0.1% benzaldehyde and shaking gently for 12 h. The unbound aptamer and residual benzaldehyde were removed by rinsing with ultrapure water. The functionalized PAA membrane with aptamer immobilized on the inner wall of PAA nanochannels (PAA/aptamer) was thus obtained and stored in Tris buffer solution (10 mM, pH 7.4) at 4 °C. Conformational Switching of the Aptamer in the Nanochannels and Regeneration of the Nanodevice. The conformational switching of the aptamer in the nanochannels was carried out by immersing the PAA/aptamer into 1 mL of Tris buffer solution (10 mM, pH 7.4) containing various concentrations of K+ or ATP and shaking gently at room temperature for 1 h. The residual K+ or ATP was removed by washing with Tris buffer solution thoroughly. The regeneration of the nanodevice could be achieved by incubating the used PAA/aptamer in 10 mL of Tris buffer solution (10 mM, pH 7.4) at 90 °C with gentle shaking for 10 min and then rinsing with Tris buffer solution. This procedure was repeated once to ensure that the K+ or ATP was thoroughly removed and that the aptamer was completely melted. Electrochemical Measurements. All electrochemical measurements were performed with a homemade nanodevice (Scheme S1). Briefly, a platinum disk was placed on a conductive copper pedestal as a working electrode. Then, the PAA membrane was put on the platinum disk. After that, an insulating block (i.e., poly(methyl methacrylate), PMMA) containing a cell for 2 mL of electrolyte (Tris buffer solution, 10 mM, pH 7.4) was placed onto the PAA membrane, with a silicone O-ring between them to prevent any liquid leakage. A CHI 660C electrochemical workstation (Chenhua Instrument, Shanghai, China) was used for all electrochemical measurements. A Pt working electrode under the PAA membrane as
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RESULTS AND DISCUSSION Conformational Switching of the Aptamers. The conformational switching of the aptamers was determined by CD spectroscopy measurements. As shown in Figure 1A, when
Figure 1. CD spectra of the G-quadruplex conformations at different concentrations of target molecules. (A) K+ aptamer at 0, 10 μM, 100 μM, and 1 mM concentrations of K+. (B) ATP aptamer at 0, 100 μM, and 1 mM concentrations of ATP.
the concentration of K+ increased from 0 to 1 mM, the CD spectrum of the K+ aptamer showed two positive peaks near 295 and 255 nm, a crossover around 265 nm, and a negative peak near 235 nm. These data indicated that the aptamer folded into two coexisting G4 conformations: a hybrid conformation and an antiparallel conformation,41,42 as shown in Scheme 2A,B. Furthermore, it was observed that the intensity of the positive peak near 295 nm increased with the concentration of K+, which suggested that the major structure of the K+ aptamer was the G4 conformation within the concentration ranging from 0 to 1 mM. These observations demonstrated that the conformational switching of the K+ aptamer in solution was intimately dependent on the concentration of K+. On the other hand, as shown in Figure 1B, the CD spectrum of the ATP 10743
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Scheme 2. G-Quadruplex Conformations of the Aptamersa
a
(A) Hybrid conformation of the K+ aptamer, (B) anti-parallel conformation of the K+ aptamer, and (C) anti-parallel conformation of the ATP aptamer.
Specifically, as shown in Figure 2A, the steady-state current for the K+ aptamer-modified PAA nanochannels (PAA/K+
aptamer without the addition of ATP showed a positive peak near 265 nm and a negative peak near 240 nm. It was also observed that both the positive and negative peaks exhibited a red shift upon the addition of ATP. When the concentration of ATP increased to 1 mM, a positive peak near 285 nm and a negative peak near 255 nm were observed, corresponding to an antiparallel G4 structure induced by ATP,43 as shown in Scheme 2C. These observations demonstrated that the conformational switching of the ATP aptamer in solution was also dependent on the concentration of ATP. It should be noted here that concentrations of ATP above 1 mM caused an overflow for the ultraviolet absorption, which made the CD spectroscopy measurement unstable in this study. Conformational Switching of Aptamers Confined in Nanochannels. A nanochannel-based electrochemical method was applied to study the relationship between the conformational switching of various aptamers and the concentration of their target molecules. As shown in Scheme 1, the aptamer assembled onto the inner wall of the nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. When the target molecule (K+ or ATP) was introduced into the nanochannels, it coupled with the aptamer to form a G4 structure. This resulted in a decrease of the free transport region for the indicator molecules, especially when the size of the biomolecules were comparable with the pore diameter of the nanochannels.18,38 Subsequently, there was a reduced flux of indicator molecules inside the nanochannels due to the increased steric hindrance. It was also reported that single-stranded DNA would lie on the inner wall of the nanochannels, which had a slight steric hindrance effect on the flux of indicator molecules because its size was much smaller than the pore diameter of the nanochannels.19 Therefore, in our case, the aptamer on the inner wall of the nanochannels without target molecules would have a slight influence on the flux of indicator molecules (HQ). Once the target molecule was introduced and the aptamer was folded into a relatively rigid G4 structure, the steric hindrance inside the nanochannels could be more profound. This would greatly reduce the flux of indicator molecules. Because the electrochemical signal (i.e., the steady-state anodic current of the indicator molecules) was in direct proportion to the steadystate flux of indicator molecules, the electrochemical signal should be reduced significantly here.
Figure 2. Time-course curves of electrochemical current for (A) bare PAA (a), bare PAA with 1 mM K+ (b), K+ aptamer-modified PAA (c), and K+ aptamer-modified PAA with 1 mM K+ (d) and (B) bare PAA (a), bare PAA with 10 mM ATP (b), ATP aptamer-modified PAA (c), and ATP aptamer-modified PAA with 10 mM ATP (d). The pore diameter of the PAA nanochannels was 25 nm.
aptamer) was 3.91 μA, and the current decreased to 3.03 μA after the PAA/K+ aptamer was incubated with 1 mM K+ (PAA/ K+ aptamer/K+). This result was consistent with the discussion above, which indicated that the G4 structure of the K+ aptamer was formed in the nanochannels. The control experiments showed that the current for bare PAA nanochannels (PAA) was 4.09 μA, only slightly larger than that for the PAA/K+ aptamer, confirming that the aptamer on the inner wall of the nanochannels without target molecules had a slight influence on the flux of indicator molecules. Additionally, it was observed that the current for bare PAA nanochannels incubated with 1 mM K+ (PAA/K+) was 4.06 μA, which was nearly the same as that at the bare PAA nanochannels. This fact demonstrated that the current drop between PAA/K+ aptamer and PAA/K+ aptamer/K+ was induced by conformational switching of the K+ aptamer in the nanochannels. On the other hand, as shown in Figure 2B, the steady-state currents for the ATP aptamermodified PAA nanochannels (PAA/ATP aptamer), PAA/ATP aptamer incubated with 10 mM ATP (PAA/ATP aptamer/ ATP), bare PAA nanochannels (PAA), and bare PAA nanochannels incubated with 10 mM ATP (PAA/ATP) were 3.79, 2.88, 4.00, and 3.99 μA, respectively. These results were quite similar to those of the K+ aptamers. Therefore, the conformational switching of various aptamers in the nanochannels occurs in the presence of their target molecules, which 10744
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and 100 μM for aptamer-based colorimetric45 methods but lower than the limit of 75 nM for the aptamer-based fluorescent method.46 In the case of the ATP aptamer, as shown in Figure 3B, when the concentration of ATP increased from 0 to 10 mM, the steady-state current dropped from 3.79 to 2.88 μA. It could be inferred that when the concentration of ATP was lower than 50 μM the current drop was negligible. When the concentration of ATP was higher than 5 mM, the current drop reached a maximum value. This suggested the the affinity between ATP and the ATP aptamer was lower than that between K+ and the K+ aptamer. On the other hand, as shown in Figure 3D, when the concentration of ATP increased from 0 to 10 mM, the value of D increased remarkably, which suggested an increasing steric hindrance in the nanochannels. This result indicated that the conformational switching of the ATP aptamer in the nanochannels was also intimately dependent on the concentration of ATP, which further demonstrated that the conformational switching of various aptamers in the nanochannels was dependent on the concentration of their target molecules, confirming the universality of this nanodevice. The inset plot in Figure 3D shows a linear relationship between D and the concentration of ATP (c), with a linear equation of D = −0.130 + 0.0931 log(c/μM) and a correlation coefficient of 0.991. The linear range for ATP concentration was from 50 μM to 10 mM, and the detection limit was 5 μM at a signal-to-noise ratio of 3, which was higher than the limit of 10 μM for the aptamer-based fluorescent method47 but lower than the limit of 1 nM for the aptamer-based luminescent method.48 In addition, when the concentration of the target was high enough, the corresponding aptamer in the PAA nanochannels would be almost entirely folded into the G4 structure. By comparing Figure 3, panels C and D, it could be concluded that when the aptamers in the PAA nanochannels were almost entirely folded into their G4 structures the value of D for the ATP aptamer was larger than that for the K+ aptamer. This indicated that the steric hindrance from the G4 structure of the ATP aptamer in the PAA nanochannels was a bit more profound than that of the K+ aptamer. This fact directly demonstrated that the dimension of the G4 structure of the ATP aptamer was larger than that of the G4 structure of the K+ aptamer. Impacts of Pore Diameter, Ionic Strength, and pH on the Nanodevice. The steady-state flux of indicator molecules was controlled by the free transport region for indicator molecules in the nanochannels. When the steric hindrance was constant in the nanochannels, the pore diameter of the nanochannels was a predominant factor for the steady-state flux of indicator molecules and thus the steady-state current. SEM images of the top and cross-sectional views of PAA membranes with various pore diameters are shown in Figure 4A. It could be observed that the nanochannels had uniform sizes throughout their lengths (Figure S2) and that the pore diameters of these PAA nanochannels were 25, 55, 90, and 130 nm. Figure 4B,C shows the relationship between the values of D and the pore diameters of the PAA nanochannels with the K+ aptamer and the ATP aptamer, respectively. It was obvious that in both cases, when the pore diameter of the nanochannels increased, the value of D decreased greatly. This result clearly illustrated that the effect of steric hindrance on the flux of indicator molecules in the nanochannels was reduced significantly when the pore diameter of the nanochannels increased. Therefore, the PAA nanochannels with a pore
increases the steric hindrance in the nanochannels and results in a reduced flux of indicator molecules inside the nanochannels. The applicability of this nanodevice was thus demonstrated. Electrochemical Detection of the Target Molecules of Aptamers. The CD spectroscopy measurements suggested that the conformational switching of the aptamers was dependent on the concentration of the target molecules. Thus, the nanochannel-based electrochemical platform could be applied to detect the concentration of the target. As shown in Figure 3A, when the concentration of K+ increased from 0 to
Figure 3. Time-course curves of current for (A) K+ aptamer-modified PAA at K+ concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, and 1 mM and (B) ATP aptamer-modified PAA at ATP concentrations of 0, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM. (C) Calibration curves of the current drop ratio versus K+ concentration for K+ aptamer-modified PAA and (D) calibration curves of the current drop ratio versus ATP concentrations for ATP aptamer-modified PAA. Insets: linear plots for (C) K+ and (D) ATP determinations. The pore diameter of the PAA nanochannels was 25 nm.
1 mM, the steady-state current dropped from 3.91 to 3.03 μA. It could be inferred that when the concentration of K+ was lower than 1 μM the current drop was negligible. On the other hand, when the concentration of K+ was higher than 500 μM, the current drop reached a maximum value. Here, a parameter, D, was introduced to represent the current drop ratio, which was defined as D = 1 − I/I0, where I0 and I are the steady-state currents for the PAA/K+ aptamer and PAA/K+ aptamer/K+, respectively. As shown in Figure 3C, when the concentration of K+ increased from 0 to 1 mM, the value of D increased remarkably, which suggested an increasing steric hindrance in the nanochannels. This suggested that the conformational switching of the K+ aptamer in the nanochannels was intimately dependent on the concentration of K+. In short, the higher the concentration of K+, the more conformational switching of the K+ aptamer that occurs. The inset plot in Figure 3C shows a linear relationship between D and the concentration of K+ (c), with a linear equation of D = −0.0388 + 0.105 log(c/μM) and a correlation coefficient of 0.997. The linear range for K+ concentration was from 5 to 200 μM, and the detection limit was 0.4 μM at a signal-to-noise ratio of 3, which was higher than the limit of 15 μM for aptamer-based electrochemical44 10745
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Figure 4. (A) SEM images for the top (top) and cross-sectional (bottom) views of PAA membranes with various pore diameters: 25, 55, 90, and 130 nm (from left to right). The impact of pore diameter on the current drop ratio for (B) K+ aptamer-modified PAA with 1 mM K+ and (C) ATP aptamer-modified PAA with 10 mM ATP. The scale bar is 200 nm.
This suggested that the commonly used pH for biosensors had an acceptable impact on the PAA/ATP aptamer. These results demonstrated that this nanodevice was insensitive to the common ionic strengths and pH values used for biosensors, which showed the practicality of the nanodevice. Reproducibility, Life Span, and Selectivity of the Nanodevice. The reproducibility of this approach was evaluated by investigating five individual PAA membranes for the K+ aptamer and the ATP aptamer, respectively. The steadystate currents not only for the five PAA/K+ aptamer membranes but also for the corresponding five PAA/K+ aptamer/K+ membranes were similar to each other (Figure S3A). The slight differences between them may be a result of the inherent differences between the PAA membranes. Figure S4A illustrates the values of D from the five individual PAA membranes for the K+ aptamer, which were found to be close to each other. Additionally, the relative standard deviation (RSD) was calculated to be 3.5%. In the case of the ATP aptamer, Figures S3B and S4B illustrate similar results to those for the K+ aptamer, and the corresponding RSD was calculated to be 5.2%. Therefore, good reproducibility of this approach was demonstrated. The life span of this nanodevice was evaluated by counting the regeneration and reuse cycles of the PAA/aptamer. In the case of the PAA/K+ aptamer, the value of D was steady in the first 4 cycles for the detection of K+, as shown in Figure S5A. It could be observed that the value of D had an inconspicuous decrease in the fifth cycle and a continued the decrease in the following cycles. This suggested that the regeneration and reuse of the PAA/K+ aptamer were acceptable for 4 cycles. In the case of the PAA/ATP aptamer, as shown in Figure S5B, the value of D was also steady in the first 4 cycles for the detection of ATP. It was obvious that the value of D decreased starting with the fifth cycle, which means that the regeneration and reuse of the PAA/ATP aptamer were also 4 cycles. These results
diameter of 25 nm were most suitable for the electrochemical measurements in this study. On the other hand, the impact of ionic strength and pH on the nanodevice was also investigated. In order to obtain wide ranges of ionic strength and pH, two series of phosphate buffer solutions (PBS) with different ionic strengths and pH values were used instead of the Tris buffer solution. It was reported that the K+ aptamer could be folded into the G4 structure by some of the alkali metal ions (e.g., Na+ or K+ in PBS) at high concentrations.49 Thus, the PAA/ATP aptamer was typically investigated under conditions of different ionic strength and pH. Figure 5A shows the relationship between the value of D
Figure 5. Impact of (A) ionic strength and (B) pH on the current drop ratio for the ATP aptamer-modified PAA with 10 mM ATP. The pore diameter of the PAA nanochannels was 25 nm.
and the ionic strength. It was clear that the value of D was stable as the ionic strength ranged from 1 mM to 1 M, which indicated that the commonly used ionic strength for biosensors had little impact on the PAA/ATP aptamer. Additionally, Figure 5B shows the relationship between the value of D and the pH. It could be observed that the value of D was stable as the pH varied from 6 to 8 and that it was slightly decreased when the pH decreased from 6 to 5 or increased from 8 to 9. 10746
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demonstrated that the reliable life span of this nanodevice is 4 cycles. The selectivity of this approach to detect the target molecules was evaluated by investigating the electrochemical signals of PAA/aptamers to the analogues of their target molecules. In the case of the K+ aptamer, 1 mM of five analogues (i.e., Li+, Na+, K+, Rb+, and Cs+) was applied. The value of D for K+ (21.8%) was remarkable, whereas the values of D for the other analogues (Li+ 0.2%, Na+ 0.8%, Rb+ 0.9%, and Cs+ 0.5%) were almost negligible (Figure S6A). This suggested that at the present concentration only K+ could fold the K+ aptamer into the G4 structure in the PAA nanochannels. In the case of the ATP aptamer, 10 mM of four analogues (i.e., ATP, CTP, GTP, and UTP) was applied. Figure S6B demonstrates that the value of D for ATP (23.4%) was much larger than that for its analogues (CTP 2.5%, GTP 0.7%, and UTP 1.0%). This indicated that only ATP could fold the ATP aptamer into the G4 structure in the PAA nanochannels. These results demonstrated the significant selectivity of this approach. Furthermore, as was reported, the K+ aptamer could also be folded into the G4 structure by the analogues of K+ at high concentrations (e.g., 100 mM), especially for Na+.49 When the concentration of Na+ reached as high as 1 M, a striking current drop for the PAA/K+ aptamer occurred (Figure S7). The corresponding value of D was 22.8%, which was comparable to that for K+. Therefore, the selectivity of the PAA/K+ aptamer to detect its target molecule was acceptable when the concentrations of target molecules were relatively low.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-25-52090613. Fax: +86-25-52090618. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the Key Program (grant no. 21035002) from the National Natural Science Foundation of China, the National Natural Science Foundation of China (grant nos. 21375014 and 21175021), the Social Development Project of Suzhou (ZXY2012027), the Funding of Jiangsu Innovation Program for Graduate Education (KYLX_0163), and the Fundamental Research Funds for the Central Universities.
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REFERENCES
(1) Tamayo, J.; Kosaka, P. M.; Ruz, J. J.; San Paulo, A.; Calleja, M. Chem. Soc. Rev. 2013, 42, 1287−1311. (2) de la Escosura-Muniz, A.; Merkoci, A. ACS Nano 2012, 6, 7556− 7583. (3) Gooding, J. J. Small 2006, 2, 313−315. (4) Kirsch, J.; Siltanen, C.; Zhou, Q.; Revzin, A.; Simonian, A. Chem. Soc. Rev. 2013, 42, 8733−8768. (5) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999, 398, 686−690. (6) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001, 19, 636−639. (7) Cherf, G. M.; Lieberman, K. R.; Rashid, H.; Lam, C. E.; Karplus, K.; Akeson, M. Nat. Biotechnol. 2012, 30, 344−348. (8) Manrao, E. A.; Derrington, I. M.; Laszlo, A. H.; Langford, K. W.; Hopper, M. K.; Gillgren, N.; Pavlenok, M.; Niederweis, M.; Gundlach, J. H. Nat. Biotechnol. 2012, 30, 349−354. (9) Iqbal, S. M.; Akin, D.; Bashir, R. Nat. Nanotechnol. 2007, 2, 243− 248. (10) Hou, X.; Dong, H.; Zhu, D. B.; Jiang, L. Small 2010, 6, 361− 365. (11) Lee, W.; Ji, R.; Gosele, U.; Nielsch, K. Nat. Mater. 2006, 5, 741− 747. (12) Yang, S. Y.; Son, S.; Jang, S.; Kim, H.; Jeon, G.; Kim, W. J.; Kim, J. K. Nano Lett. 2011, 11, 1032−1035. (13) Dekker, C. Nat. Nanotechnol. 2007, 2, 209−215. (14) Kowalczyk, S. W.; Kapinos, L.; Blosser, T. R.; Magalhaes, T.; van Nies, P.; Lim, R. Y. H.; Dekker, C. Nat. Nanotechnol. 2011, 6, 433− 438. (15) Venkatesan, B. M.; Bashir, R. Nat. Nanotechnol. 2011, 6, 615− 624. (16) Wei, R. S.; Gatterdam, V.; Wieneke, R.; Tampe, R.; Rant, U. Nat. Nanotechnol. 2012, 7, 257−263. (17) Han, C. P.; Hou, X.; Zhang, H. C.; Guo, W.; Li, H. B.; Jiang, L. J. Am. Chem. Soc. 2011, 133, 7644−7647. (18) de la Escosura-Muniz, A.; Merkoçi, A. Small 2011, 7, 675−682. (19) Li, S. J.; Li, J.; Wang, K.; Wang, C.; Xu, J. J.; Chen, H. Y.; Xia, X. H.; Huo, Q. ACS Nano 2010, 4, 6417−6424. (20) Gao, H. L.; Li, C. Y.; Ma, F. X.; Wang, K.; Xu, J. J.; Chen, H. Y.; Xia, X. H. Phys. Chem. Chem. Phys. 2012, 14, 9460−9467. (21) Chen, W.; Wu, Z. Q.; Xia, X. H.; Xu, J. J.; Chen, H. Y. Angew. Chem., Int. Ed. 2010, 49, 7943−7947. (22) Chen, W.; Wu, J. S.; Xia, X. H. ACS Nano 2008, 2, 959−965. (23) Jani, A. M. M.; Kempson, I. M.; Losic, D.; Voelcker, N. H. Angew. Chem., Int. Ed. 2010, 49, 7933−7937. (24) Kim, B.; Jung, I. H.; Kang, M.; Shim, H. K.; Woo, H. Y. J. Am. Chem. Soc. 2012, 134, 3133−3138. (25) Gourine, A. V.; Llaudet, E.; Dale, N.; Spyer, K. M. Nature 2005, 436, 108−111.
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CONCLUSIONS In summary, a PAA nanochannel-based electrochemical platform was developed for the sensitive detection of K+ and ATP. It was demonstrated that both K+ and ATP G-quadruplex aptamers could be confined in PAA nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Both aptamers in the nanochannels could be folded into G4 structures in the presence of target molecules, which increased the steric hindrance in the nanochannels and resulted in a reduced flux of indicator molecules inside the nanochannels. The conformational switch of the aptamers in the PAA nanochannels was intimately dependent on the concentrations of K+ and ATP. Therefore, the quantitative detection of K+ and ATP was successfully realized with this PAA nanochannel-based electrochemical platform. The impact of pore diameter on the analyzed performances of the nanochannel-based electrochemical platform was investigated, and the reproducibility and selectivity of the nanochannel-based electrochemical platform were demonstrated. Moreover, the universality of this platform showed its potential for use the in detection of other aptamer-based analytes, which could promote the development of its use for biological detection and clinical diagnosis.
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ASSOCIATED CONTENT
S Supporting Information *
Scheme of the assembled electrochemical nanodevice, cyclic voltammogram of HQ, SEM images of PAA nanochannels, and figures for reproducibility, life span and selectivity of the nanodevice. This material is available free of charge via the Internet at http://pubs.acs.org. 10747
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(26) Kuo, H. C.; Cheng, C. F.; Clark, R. B.; Lin, J. J. C.; Lin, J. L. C.; Hoshijima, M.; Nguyen-Tran, V. T. B.; Gu, Y. S.; Ikeda, Y.; Chu, P. H.; Ross, J.; Giles, W. R.; Chien, K. R. Cell 2001, 107, 801−813. (27) Przedborski, S.; Vila, M. Clin. Neurosci. Res. 2001, 1, 407−418. (28) Thibon, A.; Pierre, V. C. J. Am. Chem. Soc. 2009, 131, 434−435. (29) Nakano, S.; Fukuda, M.; Tamura, T.; Sakaguchi, R.; Nakata, E.; Morii, T. J. Am. Chem. Soc. 2013, 135, 3465−3473. (30) Tombelli, S.; Minunni, A.; Mascini, A. Biosens. Bioelectron. 2005, 20, 2424−2434. (31) Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408− 6418. (32) Iliuk, A. B.; Hu, L. H.; Tao, W. A. Anal. Chem. 2011, 83, 4440− 4452. (33) Famulok, M.; Mayer, G. Acc. Chem. Res. 2011, 44, 1349−1358. (34) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668−698. (35) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990−17991. (36) He, F.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2005, 127, 12343−12346. (37) Xue, Q. W.; Wang, L.; Jiang, W. Chem. Commun. 2013, 49, 2640−2642. (38) Hou, X.; Guo, W.; Xia, F.; Nie, F. Q.; Dong, H.; Tian, Y.; Wen, L. P.; Wang, L.; Cao, L. X.; Yang, Y.; Xue, J. M.; Song, Y. L.; Wang, Y. G.; Liu, D. S.; Jiang, L. J. Am. Chem. Soc. 2009, 131, 7800−7805. (39) Li, S. J.; Wang, C.; Wu, Z. Q.; Xu, J. J.; Xia, X. H.; Chen, H. Y. Chem.Eur. J. 2010, 16, 10186−10194. (40) Wang, X.; Smirnov, S. ACS Nano 2009, 3, 1004−1010. (41) Rezler, E. M.; Seenisamy, J.; Bashyam, S.; Kim, M. Y.; White, E.; Wilson, W. D.; Hurley, L. H. J. Am. Chem. Soc. 2005, 127, 9439−9447. (42) Ambrus, A.; Chen, D.; Dai, J. X.; Bialis, T.; Jones, R. A.; Yang, D. Z. Nucleic Acids Res. 2006, 34, 2723−2735. (43) Zhou, Z. X.; Du, Y.; Dong, S. J. Anal. Chem. 2011, 83, 5122− 5127. (44) Radi, A. E.; O’Sullivan, C. K. Chem. Commun. 2006, 3432−3434. (45) Lee, J.; Kim, H. J.; Kim, J. J. Am. Chem. Soc. 2008, 130, 5010− 5011. (46) Huang, C. C.; Chang, H. T. Chem. Commun. 2008, 1461−1463. (47) Zhang, Z. X.; Sharon, E.; Freeman, R.; Liu, X. Q.; Willner, I. Anal. Chem. 2012, 84, 4789−4797. (48) Wang, J.; Jiang, Y. X.; Zhou, C. S.; Fang, X. H. Anal. Chem. 2005, 77, 3542−3546. (49) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286−14287.
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Quantitative Detection of Potassium Ions and Adenosine Triphosphate via a Nanochannel-Based Electrochemical Platform Coupled with G‑Quadruplex Aptamers Jiachao Yu, Linqun Zhang, Xuan Xu, and Songqin Liu* Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Suzhou Research Institute of Southeast University, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, P. R. China S Supporting Information *
ABSTRACT: The development of synthetic nanopores and nanochannels that mimick ion channels in living organisms for biosensing applications has been, and still remains, a great challenge. Although the biological applications of nanopores and nanochannels have achieved considerable development as a result of nanotechnology advancements, there are few reports of a facile way to realize those applications. Herein, a nanochannelbased electrochemical platform was developed for the quantitative detection of biorelated small molecules such as potassium ions (K+) and adenosine triphosphate (ATP) in a facile way. For this purpose, K+ or ATP G-quadruplex aptamers were covalently assembled onto the inner wall of porous anodic alumina (PAA) nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Conformational switching of the aptamers confined in the nanochannels occurs in the presence of the target molecules, resulting in increased steric hindrance in the nanochannels. Changes in steric hindrance in the nanochannels were monitored by the anodic current of indicator molecules transported through the nanochannels. As a result, quantitative detection of K+ and ATP was realized with a concentration ranging from 0.005 to 1.0 mM for K+ and 0.05 to 10.0 mM for ATP. The proposed platform displayed significant selectivity, good reproducibility, and universality. Moreover, this platform showed its potential for use in the detection of other aptamer-based analytes, which could promote its development for use in biological detection and clinical diagnosis.
R
DNA sequences and detecting proteins and other analytes.13−15 For single nanochannel-based nanodevices, the typical strategy involves the measurement of electrical conductance between two sides of the membrane, which changes based on the presence of molecules in the nanochannel.16,17 As for nanochannel array-based nanodevices, the nanochannel arrays are first modified with biorecognition molecules and are then used as a coating on the surface of conventional electrotransducers.18 By monitoring the variations in the electrochemical signals of the indicator molecules transported through the nanochannels, the presence of target molecules in the nanochannels could be determined.19 The fundamental mechanism of these nanochannel-based applications is the transport of indicator molecules or ions inside the nanochannels. Such transport is generally affected by two factors: steric hindrance and electrostatic repulsion.19 Steric hindrance is induced by the space occupied by the biomolecules inside the nanochannels.17 Because the size of the biomolecules
ecently, the development of synthetic nanopores and nanochannels that mimick the ion channels in living organisms and biosensing applications has attracted considerable attention.1−4 In living systems, the selective transportation of ions in protein-based ion channels is the source of electrical signals in nerves and muscles. This natural behavior inspired the application of nanochannels to biosensor development. The pioneer works in this field were realized by inserting a natural protein ion channel, such as α-hemolysin, into lipid bilayers to form a single biological nanochannel.5 The ionic current through such nanochannels could be altered when a target molecule appeared in the specific region of the nanochannel.6 Following this work, a number of innovative nanochannel-based biosensing systems have been developed, with special focus on DNA sequencing.7,8 Very recently, artificial single nanochannels and nanochannel arrays, which were built and embedded in mechanically and chemically robust synthetic membranes, have been prepared by utilizing various materials and technologies.9−12 The shape, size, and length of these nanochannels in membranes could be wellcontrolled during fabrication, demonstrating their remarkable promise for use in nanodevices for biosensing, e.g., monitoring © 2014 American Chemical Society
Received: July 24, 2014 Accepted: October 10, 2014 Published: October 10, 2014 10741
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Scheme 1. Functionalization of PAA Nanochannels with Aptamer and Flux of the Redox Indicator (Hydroquinone) in the Nanochannels before and after Conformational Switching of the Aptamer
bonding interactions, which are promoted by the presence of their targets.34−37 Jiang et al. reported a K+-responsive PET single nanochannel on the basis of G-quadruplex DNA conformational switching.38 In their work, G-quadruplex DNA was immobilized onto a synthetic nanopore, and it underwent a potassium-responsive conformational change that induced a change in the effective pore size. The responsiveness could be regulated by the stability of the G-quadruplex structure through adjusting the potassium concentration, which was monitored by the transmembrane ion current across the PET film. Inspired by potassium-responsive G-quadruplex DNA conformational switching in a nanopore, a nanochannel-based electrochemical platform was developed for the quantitative detection of K+ and adenosine triphosphate (ATP) in a facile way. As shown in Scheme 1, the PAA nanochannels were silanized and then modified with single-stranded K+ or ATP Gquadruplex aptamers through a Schiff reaction. When the target molecules were introduced into the nanochannels, the aptamers fold into G-quadruplex structures. Such conformational switching results in a significant increase in the steric hindrance in the nanochannels, which leads to a decrease in the flux of indicator molecules (hydroquinone, HQ) in the nanochannels. Because the flux of indicator molecules was monitored by their anodic current at the working electrode, the target molecules could be detected in a simple and rapid way.
is non-negligible compared with the pore diameter of the nanochannels, the free transport area for the indicator molecules or ions decreased. Electrostatic repulsion, however, was induced not only by the charge of the biomolecules20 but also the surface charge on the inner wall of the nanochannels.21 These charges generated an electrostatic field inside the nanochannels, which would attract or repel charged molecules and ions, and thus the transport of indicator molecules or ions was affected. On the basis of this principle, unlike stable steric hindrance, electrostatic repulsion can be influenced by the environment, such as the ionic strength and pH of the solution. Porous anodic alumina (PAA) membrane is a popular material for nanochannel-based biosensor design because of its tunable nanopore diameter, well-defined nanochannel array, robust overall structure, ease of surface functionalization, and commercial availability.22,23 For instance, Merkoçi et al.18 reported a novel PAA nanochannel-based biosensor for antigen detection. In their work, the PAA membrane was modified with antibodies, and the conductivity of the membrane toward a redox indicator was tuned by primary and secondary immunoreactions with proteins and gold nanoparticles in the nanochannels. The blockage by gold nanoparticles was enhanced by silver deposition that further decreased the diffusion of the signaling indicator through the nanochannels. The principle of such applications was based on the modulation of indicator molecules transported through the nanochannels, and their applicability depended intimately on the surface characteristics of the nanochannel’s inner walls. K+ and ATP are both essential species in the human body that play fundamental roles in various biological processes.24,25 Abnormal concentrations of both of these species are tightly associated with a series of diseases.26,27 Thus, the detection of K+ and ATP is vitally important for biochemical studies and clinic diagnosis.28,29 Aptamers are artificial DNA or RNA oligonucleotides that can bind to a wide variety of targets with high affinity and selectivity.30−33 In particular, single-stranded DNA aptamers with a guanine (G)-rich sequence can be folded into G-quadruplex structures via intramolecular hydrogen-
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EXPERIMENTAL SECTION Materials. K+ aptamer (5′-CHO-AAA AAA AAA AGG GTT AGG GTT AGG GTT AGG G-3′) and ATP aptamer (5′-CHO-AAA AAA AAA ACC TGG GGG AGT ATT GCG GAG GAA GG-3′) were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Porous anodic alumina (PAA) was purchased from Hefei Puyuan Nanotechnology Co. Ltd. (Anhui, China). (3-Aminopropyl)triethoxysilane (APTES), adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) were received from Sigma-Aldrich (Shanghai, China). 10742
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well as a Pt counter electrode and a saturated calomel electrode (SCE) over the PAA membrane formed a three-electrode electrochemical system. To illustrate the steric hindrance in PAA nanochannels before and after the conformational switching of the aptamer, the electrolyte containing 1 mM neutrally charged HQ was used. Agitation was applied to the electrolyte before the electrochemical measurement in order to form a homogeneous HQ concentration inside and outside the nanochannels. As shown in Scheme 1, the consumption of HQ at the working electrode decreased the concentration of HQ inside the nanochannels, which facilitated the diffusion of HQ molecules in the bulk electrolyte into the nanochannels. Thus, a flux of HQ in the nanochannels formed, and the diffusionlimited steady-state flux, which reflects the steric hindrance in the nanochannels, was indicated by the steady-state anodic current of HQ (Figure S1). It should be noted that the current did not reach a strict steady state in an acceptable amount of time. To balance these two factors, all measurements were stopped at 600 s, and the currents were consistently read at 600 s. CD Spectroscopy Measurements. CD spectra were collected on a Chirascan CD spectrometer (Applied Photophysics Ltd., UK). Wavelength scans were performed between 220 and 320 nm. A quartz cell with a path length of 10 mm was used. The Tris buffer solutions (10 mM, pH 7.4), containing 5 μM aptamer and various concentrations of K+ or ATP, were incubated at room temperature for 1 h before CD spectroscopy measurements.
Hydroquinone (HQ), LiCl, NaCl, KCl, RbCl, and CsCl were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and used as received. Ultrapure water (Thermo Fisher Barnstead, 18.2 MΩ) was used throughout the study. Covalent Linking of the Aptamer to PAA Nanochannels. The functionalization of PAA nanochannels with aptamer is illustrated in Scheme 1. The morphology and size of PAA membranes were characterized by field-emission scanning electron microscopy (SEM, ULTRA PLUS, ZEISS, Germany) at an acceleration voltage of 5 kV. PAA membranes with a thickness of 81 μm, a diameter of 5 mm, a geometry area of 19.6 mm2, and pore diameters of 25 ± 5, 55 ± 15, 90 ± 10, and 130 ± 20 nm were washed by ethanol and ultrapure water to remove any impurities in the nanochannels. After drying in a nitrogen flow at room temperature, the PAA membranes were immersed into 1 mL of an ethanol solution containing 5% APTES and shaken gently for 12 h to generate −NH2 groups on the inner wall of the PAA nanochannels.39 Then, the PAA membranes were washed with ethanol to remove any residual silane in the nanochannels and dried in a nitrogen flow at room temperature. After that, 10 μL of a 100 μM aptamer aqueous solution was dropped onto the surface of the PAA membrane and allowed to react for 24 h.40 It should be noted that the PAA membrane was placed on a frame that was fixed in an air-tight glass bottle with some water at the bottom. The saturated vapor pressure in the bottle prevented the 10 μL aqueous solution from evaporation over 24 h. The remaining amino groups were blocked by immersing the PAA membrane into 1 mL of ultrapure water containing 0.1% benzaldehyde and shaking gently for 12 h. The unbound aptamer and residual benzaldehyde were removed by rinsing with ultrapure water. The functionalized PAA membrane with aptamer immobilized on the inner wall of PAA nanochannels (PAA/aptamer) was thus obtained and stored in Tris buffer solution (10 mM, pH 7.4) at 4 °C. Conformational Switching of the Aptamer in the Nanochannels and Regeneration of the Nanodevice. The conformational switching of the aptamer in the nanochannels was carried out by immersing the PAA/aptamer into 1 mL of Tris buffer solution (10 mM, pH 7.4) containing various concentrations of K+ or ATP and shaking gently at room temperature for 1 h. The residual K+ or ATP was removed by washing with Tris buffer solution thoroughly. The regeneration of the nanodevice could be achieved by incubating the used PAA/aptamer in 10 mL of Tris buffer solution (10 mM, pH 7.4) at 90 °C with gentle shaking for 10 min and then rinsing with Tris buffer solution. This procedure was repeated once to ensure that the K+ or ATP was thoroughly removed and that the aptamer was completely melted. Electrochemical Measurements. All electrochemical measurements were performed with a homemade nanodevice (Scheme S1). Briefly, a platinum disk was placed on a conductive copper pedestal as a working electrode. Then, the PAA membrane was put on the platinum disk. After that, an insulating block (i.e., poly(methyl methacrylate), PMMA) containing a cell for 2 mL of electrolyte (Tris buffer solution, 10 mM, pH 7.4) was placed onto the PAA membrane, with a silicone O-ring between them to prevent any liquid leakage. A CHI 660C electrochemical workstation (Chenhua Instrument, Shanghai, China) was used for all electrochemical measurements. A Pt working electrode under the PAA membrane as
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RESULTS AND DISCUSSION Conformational Switching of the Aptamers. The conformational switching of the aptamers was determined by CD spectroscopy measurements. As shown in Figure 1A, when
Figure 1. CD spectra of the G-quadruplex conformations at different concentrations of target molecules. (A) K+ aptamer at 0, 10 μM, 100 μM, and 1 mM concentrations of K+. (B) ATP aptamer at 0, 100 μM, and 1 mM concentrations of ATP.
the concentration of K+ increased from 0 to 1 mM, the CD spectrum of the K+ aptamer showed two positive peaks near 295 and 255 nm, a crossover around 265 nm, and a negative peak near 235 nm. These data indicated that the aptamer folded into two coexisting G4 conformations: a hybrid conformation and an antiparallel conformation,41,42 as shown in Scheme 2A,B. Furthermore, it was observed that the intensity of the positive peak near 295 nm increased with the concentration of K+, which suggested that the major structure of the K+ aptamer was the G4 conformation within the concentration ranging from 0 to 1 mM. These observations demonstrated that the conformational switching of the K+ aptamer in solution was intimately dependent on the concentration of K+. On the other hand, as shown in Figure 1B, the CD spectrum of the ATP 10743
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Scheme 2. G-Quadruplex Conformations of the Aptamersa
a
(A) Hybrid conformation of the K+ aptamer, (B) anti-parallel conformation of the K+ aptamer, and (C) anti-parallel conformation of the ATP aptamer.
Specifically, as shown in Figure 2A, the steady-state current for the K+ aptamer-modified PAA nanochannels (PAA/K+
aptamer without the addition of ATP showed a positive peak near 265 nm and a negative peak near 240 nm. It was also observed that both the positive and negative peaks exhibited a red shift upon the addition of ATP. When the concentration of ATP increased to 1 mM, a positive peak near 285 nm and a negative peak near 255 nm were observed, corresponding to an antiparallel G4 structure induced by ATP,43 as shown in Scheme 2C. These observations demonstrated that the conformational switching of the ATP aptamer in solution was also dependent on the concentration of ATP. It should be noted here that concentrations of ATP above 1 mM caused an overflow for the ultraviolet absorption, which made the CD spectroscopy measurement unstable in this study. Conformational Switching of Aptamers Confined in Nanochannels. A nanochannel-based electrochemical method was applied to study the relationship between the conformational switching of various aptamers and the concentration of their target molecules. As shown in Scheme 1, the aptamer assembled onto the inner wall of the nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. When the target molecule (K+ or ATP) was introduced into the nanochannels, it coupled with the aptamer to form a G4 structure. This resulted in a decrease of the free transport region for the indicator molecules, especially when the size of the biomolecules were comparable with the pore diameter of the nanochannels.18,38 Subsequently, there was a reduced flux of indicator molecules inside the nanochannels due to the increased steric hindrance. It was also reported that single-stranded DNA would lie on the inner wall of the nanochannels, which had a slight steric hindrance effect on the flux of indicator molecules because its size was much smaller than the pore diameter of the nanochannels.19 Therefore, in our case, the aptamer on the inner wall of the nanochannels without target molecules would have a slight influence on the flux of indicator molecules (HQ). Once the target molecule was introduced and the aptamer was folded into a relatively rigid G4 structure, the steric hindrance inside the nanochannels could be more profound. This would greatly reduce the flux of indicator molecules. Because the electrochemical signal (i.e., the steady-state anodic current of the indicator molecules) was in direct proportion to the steadystate flux of indicator molecules, the electrochemical signal should be reduced significantly here.
Figure 2. Time-course curves of electrochemical current for (A) bare PAA (a), bare PAA with 1 mM K+ (b), K+ aptamer-modified PAA (c), and K+ aptamer-modified PAA with 1 mM K+ (d) and (B) bare PAA (a), bare PAA with 10 mM ATP (b), ATP aptamer-modified PAA (c), and ATP aptamer-modified PAA with 10 mM ATP (d). The pore diameter of the PAA nanochannels was 25 nm.
aptamer) was 3.91 μA, and the current decreased to 3.03 μA after the PAA/K+ aptamer was incubated with 1 mM K+ (PAA/ K+ aptamer/K+). This result was consistent with the discussion above, which indicated that the G4 structure of the K+ aptamer was formed in the nanochannels. The control experiments showed that the current for bare PAA nanochannels (PAA) was 4.09 μA, only slightly larger than that for the PAA/K+ aptamer, confirming that the aptamer on the inner wall of the nanochannels without target molecules had a slight influence on the flux of indicator molecules. Additionally, it was observed that the current for bare PAA nanochannels incubated with 1 mM K+ (PAA/K+) was 4.06 μA, which was nearly the same as that at the bare PAA nanochannels. This fact demonstrated that the current drop between PAA/K+ aptamer and PAA/K+ aptamer/K+ was induced by conformational switching of the K+ aptamer in the nanochannels. On the other hand, as shown in Figure 2B, the steady-state currents for the ATP aptamermodified PAA nanochannels (PAA/ATP aptamer), PAA/ATP aptamer incubated with 10 mM ATP (PAA/ATP aptamer/ ATP), bare PAA nanochannels (PAA), and bare PAA nanochannels incubated with 10 mM ATP (PAA/ATP) were 3.79, 2.88, 4.00, and 3.99 μA, respectively. These results were quite similar to those of the K+ aptamers. Therefore, the conformational switching of various aptamers in the nanochannels occurs in the presence of their target molecules, which 10744
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and 100 μM for aptamer-based colorimetric45 methods but lower than the limit of 75 nM for the aptamer-based fluorescent method.46 In the case of the ATP aptamer, as shown in Figure 3B, when the concentration of ATP increased from 0 to 10 mM, the steady-state current dropped from 3.79 to 2.88 μA. It could be inferred that when the concentration of ATP was lower than 50 μM the current drop was negligible. When the concentration of ATP was higher than 5 mM, the current drop reached a maximum value. This suggested the the affinity between ATP and the ATP aptamer was lower than that between K+ and the K+ aptamer. On the other hand, as shown in Figure 3D, when the concentration of ATP increased from 0 to 10 mM, the value of D increased remarkably, which suggested an increasing steric hindrance in the nanochannels. This result indicated that the conformational switching of the ATP aptamer in the nanochannels was also intimately dependent on the concentration of ATP, which further demonstrated that the conformational switching of various aptamers in the nanochannels was dependent on the concentration of their target molecules, confirming the universality of this nanodevice. The inset plot in Figure 3D shows a linear relationship between D and the concentration of ATP (c), with a linear equation of D = −0.130 + 0.0931 log(c/μM) and a correlation coefficient of 0.991. The linear range for ATP concentration was from 50 μM to 10 mM, and the detection limit was 5 μM at a signal-to-noise ratio of 3, which was higher than the limit of 10 μM for the aptamer-based fluorescent method47 but lower than the limit of 1 nM for the aptamer-based luminescent method.48 In addition, when the concentration of the target was high enough, the corresponding aptamer in the PAA nanochannels would be almost entirely folded into the G4 structure. By comparing Figure 3, panels C and D, it could be concluded that when the aptamers in the PAA nanochannels were almost entirely folded into their G4 structures the value of D for the ATP aptamer was larger than that for the K+ aptamer. This indicated that the steric hindrance from the G4 structure of the ATP aptamer in the PAA nanochannels was a bit more profound than that of the K+ aptamer. This fact directly demonstrated that the dimension of the G4 structure of the ATP aptamer was larger than that of the G4 structure of the K+ aptamer. Impacts of Pore Diameter, Ionic Strength, and pH on the Nanodevice. The steady-state flux of indicator molecules was controlled by the free transport region for indicator molecules in the nanochannels. When the steric hindrance was constant in the nanochannels, the pore diameter of the nanochannels was a predominant factor for the steady-state flux of indicator molecules and thus the steady-state current. SEM images of the top and cross-sectional views of PAA membranes with various pore diameters are shown in Figure 4A. It could be observed that the nanochannels had uniform sizes throughout their lengths (Figure S2) and that the pore diameters of these PAA nanochannels were 25, 55, 90, and 130 nm. Figure 4B,C shows the relationship between the values of D and the pore diameters of the PAA nanochannels with the K+ aptamer and the ATP aptamer, respectively. It was obvious that in both cases, when the pore diameter of the nanochannels increased, the value of D decreased greatly. This result clearly illustrated that the effect of steric hindrance on the flux of indicator molecules in the nanochannels was reduced significantly when the pore diameter of the nanochannels increased. Therefore, the PAA nanochannels with a pore
increases the steric hindrance in the nanochannels and results in a reduced flux of indicator molecules inside the nanochannels. The applicability of this nanodevice was thus demonstrated. Electrochemical Detection of the Target Molecules of Aptamers. The CD spectroscopy measurements suggested that the conformational switching of the aptamers was dependent on the concentration of the target molecules. Thus, the nanochannel-based electrochemical platform could be applied to detect the concentration of the target. As shown in Figure 3A, when the concentration of K+ increased from 0 to
Figure 3. Time-course curves of current for (A) K+ aptamer-modified PAA at K+ concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, and 1 mM and (B) ATP aptamer-modified PAA at ATP concentrations of 0, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM. (C) Calibration curves of the current drop ratio versus K+ concentration for K+ aptamer-modified PAA and (D) calibration curves of the current drop ratio versus ATP concentrations for ATP aptamer-modified PAA. Insets: linear plots for (C) K+ and (D) ATP determinations. The pore diameter of the PAA nanochannels was 25 nm.
1 mM, the steady-state current dropped from 3.91 to 3.03 μA. It could be inferred that when the concentration of K+ was lower than 1 μM the current drop was negligible. On the other hand, when the concentration of K+ was higher than 500 μM, the current drop reached a maximum value. Here, a parameter, D, was introduced to represent the current drop ratio, which was defined as D = 1 − I/I0, where I0 and I are the steady-state currents for the PAA/K+ aptamer and PAA/K+ aptamer/K+, respectively. As shown in Figure 3C, when the concentration of K+ increased from 0 to 1 mM, the value of D increased remarkably, which suggested an increasing steric hindrance in the nanochannels. This suggested that the conformational switching of the K+ aptamer in the nanochannels was intimately dependent on the concentration of K+. In short, the higher the concentration of K+, the more conformational switching of the K+ aptamer that occurs. The inset plot in Figure 3C shows a linear relationship between D and the concentration of K+ (c), with a linear equation of D = −0.0388 + 0.105 log(c/μM) and a correlation coefficient of 0.997. The linear range for K+ concentration was from 5 to 200 μM, and the detection limit was 0.4 μM at a signal-to-noise ratio of 3, which was higher than the limit of 15 μM for aptamer-based electrochemical44 10745
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Figure 4. (A) SEM images for the top (top) and cross-sectional (bottom) views of PAA membranes with various pore diameters: 25, 55, 90, and 130 nm (from left to right). The impact of pore diameter on the current drop ratio for (B) K+ aptamer-modified PAA with 1 mM K+ and (C) ATP aptamer-modified PAA with 10 mM ATP. The scale bar is 200 nm.
This suggested that the commonly used pH for biosensors had an acceptable impact on the PAA/ATP aptamer. These results demonstrated that this nanodevice was insensitive to the common ionic strengths and pH values used for biosensors, which showed the practicality of the nanodevice. Reproducibility, Life Span, and Selectivity of the Nanodevice. The reproducibility of this approach was evaluated by investigating five individual PAA membranes for the K+ aptamer and the ATP aptamer, respectively. The steadystate currents not only for the five PAA/K+ aptamer membranes but also for the corresponding five PAA/K+ aptamer/K+ membranes were similar to each other (Figure S3A). The slight differences between them may be a result of the inherent differences between the PAA membranes. Figure S4A illustrates the values of D from the five individual PAA membranes for the K+ aptamer, which were found to be close to each other. Additionally, the relative standard deviation (RSD) was calculated to be 3.5%. In the case of the ATP aptamer, Figures S3B and S4B illustrate similar results to those for the K+ aptamer, and the corresponding RSD was calculated to be 5.2%. Therefore, good reproducibility of this approach was demonstrated. The life span of this nanodevice was evaluated by counting the regeneration and reuse cycles of the PAA/aptamer. In the case of the PAA/K+ aptamer, the value of D was steady in the first 4 cycles for the detection of K+, as shown in Figure S5A. It could be observed that the value of D had an inconspicuous decrease in the fifth cycle and a continued the decrease in the following cycles. This suggested that the regeneration and reuse of the PAA/K+ aptamer were acceptable for 4 cycles. In the case of the PAA/ATP aptamer, as shown in Figure S5B, the value of D was also steady in the first 4 cycles for the detection of ATP. It was obvious that the value of D decreased starting with the fifth cycle, which means that the regeneration and reuse of the PAA/ATP aptamer were also 4 cycles. These results
diameter of 25 nm were most suitable for the electrochemical measurements in this study. On the other hand, the impact of ionic strength and pH on the nanodevice was also investigated. In order to obtain wide ranges of ionic strength and pH, two series of phosphate buffer solutions (PBS) with different ionic strengths and pH values were used instead of the Tris buffer solution. It was reported that the K+ aptamer could be folded into the G4 structure by some of the alkali metal ions (e.g., Na+ or K+ in PBS) at high concentrations.49 Thus, the PAA/ATP aptamer was typically investigated under conditions of different ionic strength and pH. Figure 5A shows the relationship between the value of D
Figure 5. Impact of (A) ionic strength and (B) pH on the current drop ratio for the ATP aptamer-modified PAA with 10 mM ATP. The pore diameter of the PAA nanochannels was 25 nm.
and the ionic strength. It was clear that the value of D was stable as the ionic strength ranged from 1 mM to 1 M, which indicated that the commonly used ionic strength for biosensors had little impact on the PAA/ATP aptamer. Additionally, Figure 5B shows the relationship between the value of D and the pH. It could be observed that the value of D was stable as the pH varied from 6 to 8 and that it was slightly decreased when the pH decreased from 6 to 5 or increased from 8 to 9. 10746
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demonstrated that the reliable life span of this nanodevice is 4 cycles. The selectivity of this approach to detect the target molecules was evaluated by investigating the electrochemical signals of PAA/aptamers to the analogues of their target molecules. In the case of the K+ aptamer, 1 mM of five analogues (i.e., Li+, Na+, K+, Rb+, and Cs+) was applied. The value of D for K+ (21.8%) was remarkable, whereas the values of D for the other analogues (Li+ 0.2%, Na+ 0.8%, Rb+ 0.9%, and Cs+ 0.5%) were almost negligible (Figure S6A). This suggested that at the present concentration only K+ could fold the K+ aptamer into the G4 structure in the PAA nanochannels. In the case of the ATP aptamer, 10 mM of four analogues (i.e., ATP, CTP, GTP, and UTP) was applied. Figure S6B demonstrates that the value of D for ATP (23.4%) was much larger than that for its analogues (CTP 2.5%, GTP 0.7%, and UTP 1.0%). This indicated that only ATP could fold the ATP aptamer into the G4 structure in the PAA nanochannels. These results demonstrated the significant selectivity of this approach. Furthermore, as was reported, the K+ aptamer could also be folded into the G4 structure by the analogues of K+ at high concentrations (e.g., 100 mM), especially for Na+.49 When the concentration of Na+ reached as high as 1 M, a striking current drop for the PAA/K+ aptamer occurred (Figure S7). The corresponding value of D was 22.8%, which was comparable to that for K+. Therefore, the selectivity of the PAA/K+ aptamer to detect its target molecule was acceptable when the concentrations of target molecules were relatively low.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-25-52090613. Fax: +86-25-52090618. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the Key Program (grant no. 21035002) from the National Natural Science Foundation of China, the National Natural Science Foundation of China (grant nos. 21375014 and 21175021), the Social Development Project of Suzhou (ZXY2012027), the Funding of Jiangsu Innovation Program for Graduate Education (KYLX_0163), and the Fundamental Research Funds for the Central Universities.
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CONCLUSIONS In summary, a PAA nanochannel-based electrochemical platform was developed for the sensitive detection of K+ and ATP. It was demonstrated that both K+ and ATP G-quadruplex aptamers could be confined in PAA nanochannels through a Schiff reaction between −CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Both aptamers in the nanochannels could be folded into G4 structures in the presence of target molecules, which increased the steric hindrance in the nanochannels and resulted in a reduced flux of indicator molecules inside the nanochannels. The conformational switch of the aptamers in the PAA nanochannels was intimately dependent on the concentrations of K+ and ATP. Therefore, the quantitative detection of K+ and ATP was successfully realized with this PAA nanochannel-based electrochemical platform. The impact of pore diameter on the analyzed performances of the nanochannel-based electrochemical platform was investigated, and the reproducibility and selectivity of the nanochannel-based electrochemical platform were demonstrated. Moreover, the universality of this platform showed its potential for use the in detection of other aptamer-based analytes, which could promote the development of its use for biological detection and clinical diagnosis.
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Article
ASSOCIATED CONTENT
S Supporting Information *
Scheme of the assembled electrochemical nanodevice, cyclic voltammogram of HQ, SEM images of PAA nanochannels, and figures for reproducibility, life span and selectivity of the nanodevice. This material is available free of charge via the Internet at http://pubs.acs.org. 10747
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