Research Article www.acsami.org
Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization Wenxiao Wang,† Lei Ge,† Ximei Sun, Ting Hou, and Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, People’s Republic of China
Downloaded via UNIV OF TEXAS AT EL PASO on October 30, 2018 at 15:13:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition−polymerization cycle. Herein, the design and signaling principle of aptamerswitched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics. KEYWORDS: homogeneous electrochemical assay, aptasensor, graphene, label-free, electrochemical impedance, DNA polymerization
■
homogeneous fluorescent biosensors for the detection of metal ions,19−21 small molecules,22−24 nucleic acids,25,26 enzyme activity,27,28 and proteins.29 Compared with fluorescent methods, electrochemical strategies, which also have attracted significant attention as powerful bioanalytical tools, promise the rapid and simple detection of biomolecules with high portability and affordability.30−35 Moreover, electrochemical biosensors provide a platform that can be easily interfaced with electronics for scale-up, miniaturized, and multiplexed biomarker detection,36−38 which are desirable for point-of-care and make them potentially great in disease diagnosis/management and for conducting fundamental biological studies. However, most electrochemical
INTRODUCTION
As the newest member of the carbon materials family, graphene has attracted increasing fundamental and practical research interest in nanoelectronics,1,2 biosensors,3,4 nanomedicine,5 and nanocomposites6,7 due to its extraordinary optical, mechanical, thermal, and electronic properties,8−12 as well as excellent biocompatibility. It has been reported that graphene shows much higher affinity toward nonstructured single-stranded DNA (ssDNA) due to the strong π−π stacking interaction between the hexagonal cells of graphene and the ring structures in nucleobases of ssDNA, while its interaction with doublestranded (ds) DNA, which effectively shields its nucleobases in the helical structure, is disfavored.13−17 In addition to this phenomenon, strong quenching of fluorescence from the adjacent fluorophore on graphene has also been observed.18 On the basis of the aforementioned properties, graphene has emerged as a promising platform in constructing DNA-based © 2015 American Chemical Society
Received: October 19, 2015 Accepted: December 10, 2015 Published: December 10, 2015 28566
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces
adsorbed on graphene/GCE, leading to high Ret response. The recognition of the aptamer to target switches on the hybridization between the HP-probe and SS-probe and then primes the BDP simultaneously in the presence of polymerase, which generates a long dsDNA duplex and, thus, decreases Ret response. More importantly, this BDP reaction could displace the target from aptamer to trigger the next recognitionpolymerization circularly. Thus, even one target molecule present in samples is able to induce obvious Ret change. With the use of carcinoembryonic antigen (CEA) as a proof-ofconcept target, this graphene-assisted homogeneous electrochemical impedance strategy provides an ultrasensitive electrochemical detection of CEA down to the femto-gram level. Compared to the traditional methods, the as-proposed homogeneous strategy is convenient but with high analytical performance. Notably, the isothermal and homogeneous strategy significantly reduces the analytical cost and simplifies the handling procedures, holding great potential for further applications in early clinical diagnosis.
biosensors rely on the immobilization of biorecognition probe on the electrode surface. The immobilization processes usually restrict the biorecognition probe’s configurational freedom and even change its geometry due to the steric hindrance effect of the modified electrode surface,39,40 resulting in much lower binding efficiency and rate between the targets and biorecognition probes on modified electrode surface than that in homogeneous solution, which may actually limit the routine application of electrochemical biosensors. In the past few years, DNA-based homogeneous electrochemical biosensor has been paid more and more attention,41−43 in which the hybridization between DNA structures and the recognition by the enzyme occur in the homogeneous solution without the immobilization of any biorecognition probe for target/analyte capture. The signal outputs of these homogeneous electrochemical biosensors are implemented through detecting the diffusion current of methylene blue,44,45 ferrocene,46,47 or other electroactive substances48,49 labeled DNA structures in the homogeneous reactions. Although these homogeneous electrochemical strategies enabled low detection limit, they still require the compulsory modification of an electroactive substance at its terminal. The labeling process is laborious or expensive. Thus, it is desirable to design a convenient and efficient signal output/amplification system for label-free and sensitive homogeneous electrochemical detection. Among different electrochemical transducer techniques, electrochemical impedance biosensor is a very powerful analytical tool for label-free analysis of interfacial properties related to biorecognition events occurring at the modified electrode interfaces, in addition to being nondestructive and highly sensitive.50−53 Therefore, in this contribution, taking the discrimination ability of graphene over ssDNA/dsDNA in combination with the electrochemical impedance biosensor, we developed a novel label-free homogeneous DNA-based electrochemical biosensor with ultrahigh sensitivity to overcome the aforementioned problem without immobilization of any bioprobe. Recently, graphene has been widely studied as a promising type of electrode material in the fields of electrochemical biosensing,54−59 because of its large surface area and high electrical conductivity. Thus, in this work, graphene is modified on the surface of glassy carbon electrode (GCE) and the resultant graphene/GCE is employed as the electrode substrate to construct the DNA-based homogeneous electrochemical impedance biosensor. Similar to conventional graphene-based homogeneous fluorescent DNA biosensors,60−63 ssDNA could be adsorbed on the graphene/GCE through the strong π−π stacking interaction and thus generates high electron transfer resistance (Ret), while quite low Ret is observed when the ssDNA hybridizes with its complementary ssDNA to form a dsDNA duplex, providing a high signal-tobackground ratio for the construction of sensitive homogeneous electrochemical biosensors. Signal amplification is a key component involved in the achievement of ultrasensitive electrochemical biosensor.64−66 Hence, on the basis of this graphene-assisted homogeneous electrochemical impedance biosensor, a novel aptamerswitched bidirectional DNA polymerizations (BDP) strategy, capable of both target recycling and exponential signal amplification, is compatibly developed in this study for the achievement of ultrasensitive electrochemical biosensing. In this strategy, a self-blocked DNA hairpin probe (HP-probe, containing the aptamer sequence) and an ssDNA probe (SSprobe) were ingeniously designed, both of which could be
■
EXPERIMENTAL SECTION
Chemicals and Materials. The oligonucleotides, the klenow fragment polymerase (3′ → 5′ exo-, KF polymerase), and the deoxynucleotide solution mixture (dNTPs) were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). All the synthetic oligonucleotides were HPLC purified and freeze-dried by the supplier. Their sequences were listed in Table S-1. The DNA sequences were used as provided and diluted in 10 mM phosphate-buffered saline (PBS, pH 7.4) to give stock solutions of 100 μM. Carcinoembryonic antigen (CEA) was purchased from Shanghai linc-Bio Science Co., Ltd. (Shanghai, China). SYBR Green I was obtained from Xiamen Bio-Vision Biotechnology Co., Ltd. (Xiamen, China). The monolayer graphene oxide (GO) was prepared according to Hummers’ method67 with small modification, and detailed characterizations of GO was described in Supporting Information. All reagents are analytical grade and solutions were prepared using degassed ultrapure water (specific resistance of 18 MΩcm). Apparatus. All electrochemical experiments were carried out on an Autolab electrochemical workstation (Metrohm, The Netherlands) at room temperature using a conventional three-electrode system, whereas the bare or modified GCE electrode (CHI104, Φ = 3 mm, CH Instruments, Inc., Shanghai) was used as the working electrode, a Ag/AgCl electrode and a platinum wire was employed as the reference electrode and counter electrode, respectively. The GCEs were polished stepwise with aqueous alumina slurries of 0.3 and 0.05 μm particles on microcloth, followed by rinsing thoroughly with ultrapure water (specific resistance of 18 MΩ cm) for 30 min. The cleaned GCEs were stored in ultrapure water until use. The images of gel electrophoresis were scanned by the Gel Doc XR+ Imaging System (BIO-RAD, America). The surface morphology of the electrode was investigated using a field-emission scanning electron microscope (Nova NANOSEM 450). X-ray photoelectron spectroscopy (XPS) was recorded on an ESCALAB 250Xi spectrometer (Thermo Fisher, Waltham, MA) with a monochromatic Al Kα X-ray source (1486.6 eV) operating at a pressure of 10−9 mbar. Peak positions (Binding energies) were calibrated to carbon C 1s peak (284.8 eV). Fabrication of Reduced GO Modified GCE. The preparation of reduced GO modified GCE (rGO/GCE), which employs three electro-reduction steps, is described briefly as follows. First, pnitrophenyl-modified GCE (NP/GCE) is prepared through the electro-grafting of p-nitrophenyl on the surface of cleaned bare GCE by cyclic voltammetry (CV). Prior to the electro-grafting, an aqueous solution of p-nitrophenyl diazonium salts was prepared through dissolving NaNO2 (final concentration: 5 mM) and p-nitroaniline (final concentration: 2 mM) in a 0.5 M HCl aqueous solution. The mixture was then stirred at dark in ice bath. After 10 min, the obtained 28567
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of Graphene-Assisted BDP-based Homogeneous Electrochemical Impedance Signal Amplification Strategy for Biomarker Assaya
a
The sequence of DNA molecules were described in terms of encoded domains, each of which represents a short fragment of DNA sequence. Complementarity between encoded domains is denoted by an asterisk. 8% native polyacrylamide hydrogel was prepared using 1 × tris-borateEDTA buffer (TBE, 89 mM Tris Borate, 2.0 mM EDTA, pH 8.3). The above sample mixtures were injected into the polyacrylamide hydrogel for electrophoresis. Electrophoresis was carried out at 110 V in TBE buffer for 40 min at room temperature, and stained for 20 min in a 1 × SYBR Green I solution. The resulting gel board was then illuminated with ultraviolet light and finally photographed by the gel imaging system.
solution was transferred into the electrochemical cell and the resultant p-nitrophenyl diazonium cations were electro-grafted onto the bare GCE surface through scanning two consecutive CV cycles from 0.4 V to −0.2 V (vs Ag/AgCl) at 100 mV·s−1. The obtained NP/GCE is ultrasonically washed with acetonitrile and water to eliminate nonspecific adsorption. For preparing NH2-terminated surface on GCE, four potential scans from 0.8 V to −1.0 V (vs Ag/AgCl) at 200 mV·s−1 are applied to the as-prepared NP/GCE in 0.25 M H2SO4 solution, followed by electrolysis at a potential of −0.8 V for 60 s to ensure all p-nitrophenyl groups had been electrochemically reduced to p-aminophenyl groups (AP). Then, GO sheets are deposited on the surface of AP/GCE through the immersion of AP/GCE into the solution of the prepared GO sheets (∼0.1 mg·mL−1) for 6 h. After being rinsed with water, the resulting GO/GCE is electrochemically reduced in 0.5 M NaCl solution through scanning the potential from 0.7 V to −1.2 V (vs Ag/AgCl) at 50 mV·s−1, followed by thoroughly rinsing with ultrapure water. After each step, the electrochemical behavior of the modified GCE is characterized by electrochemical impedance spectroscopy (EIS) using ferricyanide as redox probe. Homogeneous Electrochemical Impedance Detection. Prior to use, the HP-probe is refolded into a hairpin structure through heating to 95 °C for 3 min and then allowed to cool to room temperature to form the stem-loop DNA structure. Upon optimizing various conditions, the analytical procedure for this electrochemical biosensor could be briefly described as follows: In this study, the aptamer-switched BDP reactions are performed in a 45 μL homogeneous reaction mixture consisting of 1.0 μM HP-probe, 1.0 μM SS-probe, 300 μM dNTPs, 5 U KF polymerase, 1× KF polymerase buffer (10 mM Tris−HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9). After the addition of 5.0 μL CEA sample solution with different concentrations, the obtained BDP reaction mixture (50 μL) was allowed to react for 80 min at 37 °C in a constant temperature incubator. The resulting mixture (50 μL) is then transferred onto the rGO/GCE and incubated at room temperature for 20 min, followed by thoroughly rinsing with ultrapure water. Finally, the obtained DNA/rGO/GCE was introduced to electrochemical impedance measurements, all of which were performed in KCl solution (0.1 M) containing 5.0 mM [K3Fe(CN)6]/[K4Fe(CN)6] (1:1) through applying a 5 mV (peak amplitude) sinusoidal voltage perturbation to the obtained rGO/GCE at an open circuit potential over a frequency range from 100 kHz to 0.01 Hz. The electrochemical impedance data were plotted in the form of Nyquist plot. The obtained spectra were fitted using the Nova 1.1 software. Electrophoresis Demonstration of this Amplification Strategy. As a further support for this novel homogeneous aptamerswitched BDP signal amplification strategy, nondenaturating polyacrylamide gel electrophoresis (PAGE) was employed to verify and characterize its mechanism. Samples containing different DNA structures were added in 1.5 μL 6× loading buffer, respectively. An
■
RESULTS AND DISCUSSION Design Principle of this Homogeneous Electrochemical Biosensor. The design of DNA structures and working principle of the proposed homogeneous electrochemical amplification strategy based on aptamer-switched BDP were elucidated in Scheme 1, and the mechanism of which was verified by nondenaturating PAGE (Figure 1). As shown in
Figure 1. Nondenaturing PAGE verification of the aptamer-switched BDP process. (a) HP-probe; (b) SS-probe; (c) HP-probe+SS-probe; (d) HP-probe+target; (e) HP-probe+target+SS-probe; (f) HP-probe +target+SS-probe+KF+dNTPs; and (g) artificially synthesized L-DSs as DNA marker.
Scheme 1, this reaction system consists of a self-blocked HPprobe and an SS-probe. The CEA-aptamer sequence is marked as domain-a and domain-b in the HP-probe. In this study, the sequences of HP-probe and SS-probe were carefully and rationally designed to kinetically hinder any spontaneous interaction between each other in the absence of target input. As illustrated by Figure 1, both the HP-probe (lane a) and SS28568
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces
Figure 2. (A) Schematic illustration of the electrochemical fabrication procedure for rGO/GCE: (1) electro-reduction of p-nitrophenyl diazonium salts; (2) electro-reduction of p-nitrophenyl groups to p-aminophenyl groups; and (3) electro-reduction of GO to rGO. (B) The recorded electrochemical curves during the electrochemical fabrication of rGO/GCE: (1) two consecutive CVs of bare GCE in the aqueous solution of pnitrophenyl diazonium salts; (2) four consecutive CVs of the as-prepared NP/GCE in 0.25 M H2SO4 solution; and (3) two successive LSVs of GO/ GCE in 0.5 M NaCl solution.
probe (lane b) shows a single and narrow electrophoresis band, indicated that no secondary structure is observed in each rationally designed DNA sequence. Although there are complementary domain-a/a* between HP-probe and SSprobe, mixing the two DNA structures (lane c in Figure 1) in the absence of target does not produce any obvious new band, implying the very low level of spontaneous interactions between the rationally designed HP-probe and SS-probe in the absence of target. Upon target CEA introduction, the binding of the CEA molecule to the aptamer region in HP-probe results in a conformational change of HP-probe from stem-loop structure to CEA-binding structure and exposes its occluded stem region. As seen from lane d in Figure 1, the new band, appears at the shorter electrophoresis distance, mainly derives from the formation of CEA@HP complexes. This result reveals that the designed HP-probe could be opened with the recognition of target CEA. In this case, the exposed domain-a* of HP-probe could hybridize to domain-a of the SS-probe, and acts as a primer to induce enzymatic DNA polymerization reaction from its 3′-end in the presence of KF polymerase and dNTPs using SS-probe as template. At the same time, the 3′-end of the SSprobe could be also extended by the KF polymerase using the CEA-binding HP-probe as template. As expected, lane e in Figure 1 shows a new electrophoresis band at the minimum electrophoresis distance, which represents the formed CEA@ HP@SS complexes. The successful extension of both the CEA@HP and SS-probe during the BDP reactions would generate a long dsDNA duplex (denoted as L-DS in this study). As shown in Figure 1, the brightest electrophoresis band in lane f represents the generated L-DSs, which is confirmed by the electrophoresis band of artificially synthesized L-DSs (Table S1) in lane g. Furthermore, the generation of L-DSs implied that the target CEA can be displaced from the CEA-aptamer and subsequently binds to another HP-probe, triggering next recognition-polymerization reactions. This results in a target-
binding/displacement cycle-based DNA polymerization and consequently the production of numerous L-DSs. It is notable that the brightness of the electrophoresis bands corresponding to HP-probe and SS-probe in lane f declines remarkably, indicates that the successful formation of the L-DSs consumes a large amount of HP-probes and SS-probes in the homogeneous solution. Moreover, the generated L-DSs could effectively shield the ring structure of nucleobases in their long helical structures, which dramatically decrease their adsorption affinity toward rGO/GCE and thus reduce the Ret response remarkably. While in the absence of target CEA, HP-probe is incapable of hybridizing to SS-probe, inhibiting the subsequent polymerizations. In this case, all the designed HP-probes, SSprobes and dNTPs could be adsorbed on the rGO/GCE through the strong π−π stacking interaction, resulting high Ret response. Thus, by monitoring the change of Ret value, we could detect the target with high sensitivity. Characterizations of rGO/GCE. Figure 2A illustrates the electrochemical fabrication procedure for rGO/GCE, which employs three electro-reduction steps. In this work, pnitrophenyl is first electro-grafted onto the GCE surface (Figure 2A-1) by the self-limiting electro-reduction of pnitrophenyl diazonium salts. The recorded cyclic voltammograms (Figure 2B-1) for the electrochemical grafting of pnitrophenyl groups onto the surface of GCE shows a sharp irreversible reduction peak at about +0.09 V (vs Ag/AgCl) in the first cycle, which is attributed to the electro-reduction of diazonium cation.68 The disappearance of this reduction peak in the second potential cycle indicates that the electrode surface is passivated through the formation of self-limiting p-nitrophenyl film. The p-nitrophenyl groups on the GCE were then electrochemically reduced to p-aminophenyl groups (Figure 2A-2). Cyclic voltammograms for the electro-reduction of NP/ GCE are recorded in Figure 2B-2. The large irreversible reduction peak at about −0.65 V (vs Ag/AgCl) in the first scan toward the negative potentials is assigned to electro-reduction of p-nitrophenyl to p-aminophenyl.68 In the subsequent scans, 28569
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces this reduction peak drastically diminishes. After the first scanning, the reversible redox peaks that emerged at about E1/2 = +0.33 V is due to the interconversion between nitrosophenyl and hydroxyaminophenyl.68 In this work, GO sheets could be firmly attached onto the AP/GCE surface with the aid of the π−π stacking interactions between the hexagonal cells of GO and the ring of paminophenyl groups as well as the electrostatic attraction between the negatively charged GO sheets and the positively charged p-aminophenyl groups. The as-prepared GO/GCE can be electro-reduced to rGO/GCE (Figure 2A-3) using linear sweep voltammetry (LSV). During the potential scan, an intense reduction peak appeared at about −0.98 V (vs Ag/ AgCl) is observed (Figure 2B-3). It has been reported that this reduction peak comes from the reduction of the electroactive oxygen groups on the GO sheets.69 It is notable that the above reduction peak greatly diminishes in the subsequent scan, indicates that GO can be completely reduced to rGO sheets in the first scan. Compared with chemical reduction, the electroreduction approach is green and fast, and does not result in contamination of the obtained rGO. The surface morphologies of the modified electrodes were investigated by SEM. Figure S-4A,B in Supporting Information clearly shows the high magnification SEM images obtained from bare GCE surface. Uniform and slight polishing traces are obviously observed on the bare GCE surface. The surface morphology of NP/GCE (Figure S-4C) and AP/GCE (Figure S-4D), respectively, shows no significant difference from bare GCE under high magnification, indicating that the formed NP and AP thin film68 has no obvious influence on the surface morphology of electrode. In comparison to the above modified GCEs, a crumpled silk-like thin membrane, which covers the electrode surface with typical wrinkles of GO sheets, is obviously observed on GO/GCE (Figure S-4E,F). In addition, the adsorbed GO sheets on GCE exhibited transparency to the electron beam, as demonstrated by the obvious polishing traces under the GO sheets (Figure S-4F). implying that a thin GO sheets layer is obtained on the electrode surface.70 After the electro-reduction of GO sheets, the surface morphology of the rGO/GCE (Figure S-4G,H) appeared to be very similar to that of GO/GCE (Figure S-4E,F) in the SEM images, suggesting that the electro-reduction of GO sheets under the high potential of about −0.98 V (vs Ag/AgCl) does not result in significant damage to the obtained rGO sheets on electrode surface.69 The electrochemical characterizations of the modified GCE at different status are confirmed by EIS. The redox couple of [K3Fe(CN)6]/[K4Fe(CN)6], which is sensitive to surface chemistry, is used to indicate the electrochemical behaviors of the modified GCE at different stages. Figure 3A depicts the distinct impedance spectra (Nyquist diagrams) of the modified GCE at different stages and the corresponding equivalent circuit. The Nyquist plots show a semicircle at higher frequencies representing the electron-transfer-limited process and a linear part at lower frequencies resulting from the diffusion-limited process. The semicircle diameter corresponds to the interfacial electron-transfer resistance (Ret), which reflects the restricted diffusion of the redox probe accessing the electrode surface. And the Ret values can be obtained from fitted results with the equivalent circuit. It can be seen from Figure 3A that the Ret value of the bare GCE (209 Ω, curve a) increased greatly (3210 Ω, curve b) after successful electro-grafting of p-nitrophenyl film on the GCE
Figure 3. (A) Nyquist plots of the modified GCE at different stages: (a) bare GCE; (b) NP/GCE; (c) AP/GCE; (d) GO/GCE; (e) rGO/ GCE; (B) EIS response of rGO/GCE toward BDP reaction mixture, (a) without CEA; (b) with 0.5 fg·mL−1 CEA; (c) with 50.0 fg·mL−1 CEA; (d) containing SS-probe1 as a substitute of SS-probe in the presence of 50.0 fg·mL−1 CEA; (e) containing 3′-phosphorylated HPprobe and SS-probe as a substitute of nonphosphorylated HP-probe and SS-probe in the presence of 50.0 fg·mL−1 CEA. (C) EIS response of bare GCE (as a benchmark electrode) toward BDP reaction mixture (a) without and (b) with 50.0 fg·mL−1 CEA. These EIS spectra are collected in KCl solution (0.1 M) containing [K3Fe(CN)6]/[K4Fe(CN)6] (5.0 mM, 1:1) mixture as redox probe. (Insets) Equivalent circuit applied to fit each impedance spectra.
surface. This kind of electrochemical response variations mainly derives from the low conductivity of p-nitrophenyl film, which hindered the electron transfer of the redox probe on the surface of the electrode. After the electro-reduction of p-nitrophenyl film, the charge transfer between ferricyanide and GCE becomes more efficient (513 Ω curve c) probably due to the good conductivity of p-aminophenyl. Once the GO sheets are adsorbed on AP/GCE, the Ret value increases to 1780 Ω (curve d), which was mainly attributed to the high electrical resistivity 28570
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces
Figure 4. (A) Impedance responses of the proposed homogeneous electrochemical biosensor to different concentrations of CEA (from a to q: 0, 0.1, 0.2, 0.3, 0.5, 1.5, 3.0, 5.0, 15, 30, 50, 150, 300, 500, 1500, 3000, and 5000 fg·mL−1). (B) Variation of the Ret value as a function of CEA concentration and (inset) linear relationship of ΔR versus the logarithm of CEA concentration.
of GO sheets. A remarkable attenuation in Ret (120 Ω, curve e) is observed after the electro-reduction of GO to rGO, indicating an enhanced charge-carrier mobility in rGO is obtained as a result of the partially restoration of the sp2hybridized carbon network71 after electro-reduction of GO sheets, which is confirmed by the high-resolution C 1s corelevel XPS spectra in Supporting Information. These EIS measurements demonstrated the successful preparation of rGO/GCE. Feasibility of this Homogeneous Impedance Biosensor. EIS was employed to explore the feasibility of the proposed homogeneous BDP-based signal amplification strategy for this impedance biosensor using rGO/GCE as the sensing platform. As illustrated in Figure 3B, in absence of target CEA, the Ret value is about 16981 Ω (curve a). This high Ret value is considered as the direct consequence of the adsorption of HP-probes, SS-probes, and dNTPs in the BDP reaction mixture on the rGO/GCE surface through the strong π−π stacking between nucleotides and rGO. The negative charge on these DNA structures repels the negatively charged [Fe(CN)6]4−/3− anions, which retards the electron transfer between the redox probe and the electrode surface. As shown in Figure 3B, trace amounts of CEA in the BDP reaction mixture could result in a much lower impedance response (13967 Ω, curve b). Moreover, the remarkable Ret value decrease obtained from the increased concentration of CEA in the BDP reaction mixture (7412 Ω, curve c) illustrates the high sensitivity of the proposed method. To have a benchmark, the EIS profiles of the bare GCE (as a benchmark electrode) after the adsorption of BDP reaction mixture in the absence/presence of target CEA are shown in Figure 3C. It can be seen that, however, the BDP reaction mixture without target CEA results in a much higher Ret value on rGO/GCE (16981 Ω, Figure 3B, curve a) than that on bare GCE (254 Ω, Figure 3C, curve a), suggesting the higher adsorption capacity of rGO/GCE toward the BDP reaction mixture, as compared to that of the bare GCE. As shown in Figure 3C, after the adsorption of BDP reaction mixture with target CEA, the EIS profile of bare GCE (248 Ω, Figure 3C, curve b) exhibits a negligible Ret change (ΔR = 6 Ω). In contrast, the Ret value of rGO/GCE (7412 Ω, Figure 3B, curve c) toward the same amount of target CEA decreased significantly (ΔR = 9569 Ω), indicating the higher discriminability of rGO/GCE over ssDNA and dsDNA in the proposed homogeneous BDP reaction, as compared to that
of the bare GCE. Therefore, rGO/GCE can be employed as a promising sensing platform in designing, for example, highly sensitive DNA-based homogeneous electrochemical biosensors. Subsequently, we carried out a control experiment to further prove the signal amplification effect of the designed BDP strategy. Toward this goal, the SS-probe is replaced by SSprobe1 (Table S-1), which could only initiated the monodirectional DNA polymerization to release the target CEA as shown in Scheme S-1, and thus, the decrease of Ret value is greatly limited (16027 Ω, curve d). The significant difference in the Ret change between SS-probe and SS-probe1 suggests that the proposed BDP-based homogeneous electrochemical strategy in Scheme 1 possesses high amplification efficiency and works well on this rGO/GCE sensing platform. To further confirm that the Ret decrease is caused by the BDP, a control experiment is carried out. The 3′-end of both HP-probe and SS-probe are phosphorylated to inhibit polymerase elongation. It is shown that the Ret values of the reaction mixture have no obvious change by the addition of CEA (16993 Ω, curve e). Such parallel experiment indicated that the Ret decrease of the reaction system in the presence of target is due to the target induced BDP. Analytical Performance of the Impedance Biosensor. Under the optimal experimental conditions (detail optimizations are shown in Supporting Information) and according to the experimental protocol described in the Experimental Section, we then investigated the impedance response of this homogeneous impedance biosensor toward target CEA at different concentrations. As shown in Figure 4A, impedance spectra were recorded upon introduction of different concentrations of CEA. As expected, remarkable decreases in the diameter of the semicircle are clearly observed with increasing concentrations of CEA. A calibration plot based on the change in the Ret value after the target CEA addition is shown in Figure 4B. It can be clearly seen that the Ret change (ΔR, ΔR = R0 − R, where R0 and R are the Ret values of the biosensor without and with target CEA, respectively) exhibited a good linear relationship with the logarithmic (lg) value of CEA concentration in the dynamic range from 0.1 fg·mL−1 to 5.0 pg·mL−1, which can be represented by the linear regression equation ΔR(Ω) = 3257.1 × lg[cCEA(fg·mL−1)] + 3764.4 (r = 0.9978), with a directly measured detection limit of 0.1 fg· mL−1. To the best of our knowledge, the sensitivity of this proposed homogeneous impedance biosensor is comparable to or even better than most of the previously reported CEA 28571
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces biosensors. The detailed comparison of the proposed homogeneous impedance biosensor with others was illustrated in Table S-2. Such a high sensitivity is mainly attributed to the high efficiency of the developed target-recycling amplification strategy supported by the unique isothermal BDP, which triggers a large-scale consumption of HP-probe, SS-probe, and dNTPs and numerous generations of L-DSs, and thus decreases the Ret response as explained above. Moreover, the data points in the calibration curve represent six independent measurements, which show the relative standard deviations ranging from 3.16 to 4.22%, indicating good reproducibility of this homogeneous impedance biosensor. Apart from the dramatically improved sensitivity of cancer biomarker detection, our homogeneous electrochemical impedance strategy possesses some other advantages compared to the currently known assay methods for cancer biomarkers (Table S-2). First, our strategy employs aptamer as the target recognition element. Aptamer not only shows high specificity and affinity toward various targets but also possesses a series of advantages over the traditionally used antibodies, such as easy and reproducible synthesis, high resistance against denaturation, low molecular weight, programmable base sequences, and structures.72 Second, our strategy is facile and cheap because it avoids making any labels and the time- and labor-intensive labeling process at the ends or at the active sites of DNA molecules, maximally remaining the performance of the designed DNA probes. Third, our approach utilized the discrimination ability of graphene over ssDNA/dsDNA to achieve the homogeneous electrochemical measurement, which averts any complex and tedious bioprobe immobilization processes. Moreover, it should be noted that the strategy presented in this work also includes some disadvantages, similar to most DNA-based electrochemical impedance analysis methods. For example, the signal amplification circuit employs natural nuclease (KF polymerase in this work) as the catalyst, which is sensitive to environmental conditions and unstable under critical conditions (e.g., abnormal salt, temperature, and pH). Furthermore, this strategy needs a large amount of redox probe (ferricyanide in this work) dissolved in the homogeneous detection environment. Therefore, this strategy is not suitable for online and in situ detection, especially for intracellular and in vivo analysis. Selectivity and Stability of the Impedance Biosensor. To investigate the specificity of the proposed impedance sensing strategy, other protein biomarkers at the higher concentration are employed as interfering substances, such as alpha-fetoprotein (AFP), carcinoma antigen 125 (CA125), carcinoma antigen 199 (CA199). As shown in Figure 5, the contrast experiments were performed by using AFP (1.0 ng· mL−1), CA125 (1.0 ng·mL−1), and CA199 (1.0 ng·mL−1) to replace CEA (1.0 fg·mL−1), respectively. We can see that only CEA results in an evident impedance change with a sharply decreased Ret, the Ret value of AFP, CA125, and CA199 do not exhibit any obvious decrease compared with the blank. Moreover, a 0.1 fg·mL−1 CEA sample coexisted with 1.0 ng· mL−1 AFP, 1.0 ng·mL−1 CA125, and 1.0 ng·mL−1 CA199 does not exhibit obvious Ret change compared with that obtained from 0.1 fg·mL−1 CEA only. The above experiments reveal that the proposed impedance sensing strategy could offer a high selectivity for biomolecules assay, giving this impedance sensing strategy great potential for accurate early diagnosis of cancers. As an important property of the fabricated homogeneous impedance biosensor, the storage stability is necessary to be
Figure 5. Selectivity of this impedance biosensor. (A) Blank; (B) 0.1 fg·mL−1 CEA; (C) 1.0 ng·mL−1 AFP; (D) 1.0 ng·mL−1 CA125; (E) 1.0 ng·mL−1 CA199; (F) 0.1 fg·mL−1 CEA sample coexisted with 1.0 ng·mL−1 AFP, 1.0 ng·mL−1 CA125, and 1.0 ng·mL−1 CA199.
investigated. When the homogeneous impedance biosensor was stored in the refrigerator at 4 °C over 28 days, 97.5% of its initial signal response remained, suggesting a quite satisfactory storage stability. Application of this Homogeneous Electrochemical Biosensor. A preliminary application of the proposed homogeneous electrochemical biosensor was implemented in the analysis of human serum specimens, which were appropriately diluted with PBS prior to assay, to evaluate the analytical reliability and application potential of the proposed method. The assay results obtained by this method are compared with those obtained by the commercially used electrochemiluminescent method (Elecsys 2010, Roche) in cancer hospitals. The results are shown in Table 1. As can be Table 1. Assay Results of Real Human Serum by the Proposed and Reference Method CEA concn (ng·mL−1)
a
sample
proposed methoda
reference methoda
relative error (%)
1 2 3 4 5
40.08 44.39 29.11 7.75 20.09
39.27 45.18 29.75 7.48 20.68
2.05 −1.74 −2.14 3.63 −2.86
Average of 11 measurements.
seen from Table 1, the proposed biosensing strategy shows an acceptable agreement with the reference method and the relative deviation are not more than 3.63%, suggesting that it is feasible to apply the developed homogeneous electrochemical biosensor to monitoring biomarkers in human serum samples.
■
CONCLUSIONS In summary, we have demonstrated a novel homogeneous electrochemical impedance biosensing strategy for ultrasensitive and label-free target protein determination based on the difference in affinities of ssDNA and dsDNA toward graphene. A novel aptamer-switched isothermal BDP strategy has been designed to convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output. The principle of this BDP strategy was further confirmed by nondenaturating PAGE experiments. To the best of our knowledge, the described homogeneous electrochemical impedance biosensing strategy has never been reported before. The target recycling 28572
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces
(8) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Electric Field Effect in Atomically Thin Carbon Films. Science 2004, 306, 666−669. (9) Geim, A. K. Graphene: Status and Prospects. Science 2009, 324, 1530−1534. (10) Ambrosi, A.; Chua, C. K.; Bonanni, A.; Pumera, M. Electrochemistry of Graphene and Related Materials. Chem. Rev. 2014, 114, 7150−7188. (11) Loh, K. P.; Bao, Q.; Eda, G.; Chhowalla, M. Graphene Oxide as a Chemically Tunable Platform for Optical Applications. Nat. Chem. 2010, 2, 1015−1024. (12) Rao, C. N. R.; Sood, A. K.; Subrahmanyam, K. S.; Govindaraj, A. Graphene: The New Two-Dimensional Nanomaterial. Angew. Chem., Int. Ed. 2009, 48, 7752−7777. (13) Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Graphene and Graphene Oxide: Biofunctionalization and Applications in Biotechnology. Trends Biotechnol. 2011, 29, 205−212. (14) Lu, C.-H.; Yang, H.-H.; Zhu, C.-L.; Chen, X.; Chen, G.-N. A Graphene Platform for Sensing Biomolecules. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (15) He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis. Adv. Funct. Mater. 2010, 20, 453−459. (16) Tang, L.; Wang, Y.; Li, J. The Graphene/Nucleic Acid Nanobiointerface. Chem. Soc. Rev. 2015, 44, 6954−6980. (17) Tang, L.; Wang, Y.; Liu, Y.; Li, J. DNA-Directed Self-Assembly of Graphene Oxide with Applications to Ultrasensitive Oligonucleotide Assay. ACS Nano 2011, 5, 3817−3822. (18) Swathi, R. S.; Sebastian, K. L. Resonance Energy Transfer from a Dye Molecule to Graphene. J. Chem. Phys. 2008, 129, 054703. (19) Zhao, X.-H.; Kong, R.-M.; Zhang, X.-B.; Meng, H.-M.; Liu, W.N.; Tan, W.; Shen, G.-L.; Yu, R.-Q. Graphene−Dnazyme Based Biosensor for Amplified Fluorescence “Turn-on” Detection of Pb2+ with a High Selectivity. Anal. Chem. 2011, 83, 5062−5066. (20) Huang, J.; Gao, X.; Jia, J.; Kim, J.-K.; Li, Z. Graphene OxideBased Amplified Fluorescent Biosensor for Hg2+ Detection through Hybridization Chain Reactions. Anal. Chem. 2014, 86, 3209−3215. (21) Zhang, H.; Jia, S.; Lv, M.; Shi, J.; Zuo, X.; Su, S.; Wang, L.; Huang, W.; Fan, C.; Huang, Q. Size-Dependent Programming of the Dynamic Range of Graphene Oxide−DNA Interaction-Based Ion Sensors. Anal. Chem. 2014, 86, 4047−4051. (22) Wang, Y.; Li, Z.; Hu, D.; Lin, C.-T.; Li, J.; Lin, Y. Aptamer/ Graphene Oxide Nanocomplex for in Situ Molecular Probing in Living Cells. J. Am. Chem. Soc. 2010, 132, 9274−9276. (23) Lu, C.-H.; Li, J.; Lin, M.-H.; Wang, Y.-W.; Yang, H.-H.; Chen, X.; Chen, G.-N. Amplified Aptamer-Based Assay through Catalytic Recycling of the Analyte. Angew. Chem., Int. Ed. 2010, 49, 8454−8457. (24) Wang, Y.; Tang, L.; Li, Z.; Lin, Y.; Li, J. In Situ Simultaneous Monitoring of ATP and GTP Using a Graphene Oxide Nanosheet− Based Sensing Platform in Living Cells. Nat. Protoc. 2014, 9, 1944− 1955. (25) Liu, H.; Li, L.; Wang, Q.; Duan, L.; Tang, B. Graphene Fluorescence Switch-Based Cooperative Amplification: A Sensitive and Accurate Method to Detection Microrna. Anal. Chem. 2014, 86, 5487−5493. (26) Liu, H.; Li, L.; Duan, L.; Wang, X.; Xie, Y.; Tong, L.; Wang, Q.; Tang, B. High Specific and Ultrasensitive Isothermal Detection of Microrna by Padlock Probe-Based Exponential Rolling Circle Amplification. Anal. Chem. 2013, 85, 7941−7947. (27) Jang, H.; Kim, Y.-K.; Kwon, H.-M.; Yeo, W.-S.; Kim, D.-E.; Min, D.-H. A Graphene-Based Platform for the Assay of Duplex-DNA Unwinding by Helicase. Angew. Chem., Int. Ed. 2010, 49, 5703−5707. (28) Lin, L.; Liu, Y.; Zhao, X.; Li, J. Sensitive and Rapid Screening of T4 Polynucleotide Kinase Activity and Inhibition Based on Coupled Exonuclease Reaction and Graphene Oxide Platform. Anal. Chem. 2011, 83, 8396−8402.
ability of this strategy significantly amplified the electrochemical signal, and resulted in a wide linear range and femto-gram level detection of target. This assay is simple, rapid, and costefficient, and there is no need to label DNA molecule. Because numerous aptamers have been selected to bind a wide range of targets, this homogeneous electrochemical impedance biosensor holds great potential to provide a new general platform for sensitive detection of various targets and may find wide applications in the environmental and biomedical fields.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b09932. Sequences of oligonucleotides used in this work, transmission electron microscope and atomic force microscope characterizations of GO, SEM characterizations of the modified GCE as described in the text, XPS confirmations of electro-reduction of GO, supplementary Scheme S-1, detailed optimizations of the experimental conditions, and comparison of the present study with other sensing strategies. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: +86-532-86080855. Author Contributions †
W.X.W. and L.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21545005, 31501570, 21375072), Natural Science Foundation of Shandong Province, China (ZR2014BQ011), Research Foundation for Distinguished Scholars of Qingdao Agricultural University (6631115003, 663-1113311), and the Special Foundation for Taishan Scholar of Shandong Province.
■
REFERENCES
(1) Li, X.; Wang, X.; Zhang, L.; Lee, S.; Dai, H. Chemically Derived, Ultrasmooth Graphene Nanoribbon Semiconductors. Science 2008, 319, 1229−1232. (2) Roy-Mayhew, J. D.; Aksay, I. A. Graphene Materials and Their Use in Dye-Sensitized Solar Cells. Chem. Rev. 2014, 114, 6323−6348. (3) Pumera, M.; Ambrosi, A.; Bonanni, A.; Chng, E. L. K.; Poh, H. L. Graphene for Electrochemical Sensing and Biosensing. TrAC, Trends Anal. Chem. 2010, 29, 954−965. (4) Chen, D.; Feng, H.; Li, J. Graphene Oxide: Preparation, Functionalization, and Electrochemical Applications. Chem. Rev. 2012, 112, 6027−6053. (5) Mao, H. Y.; Laurent, S.; Chen, W.; Akhavan, O.; Imani, M.; Ashkarran, A. A.; Mahmoudi, M. Graphene: Promises, Facts, Opportunities, and Challenges in Nanomedicine. Chem. Rev. 2013, 113, 3407−3424. (6) Stankovich, S.; Dikin, D. A.; Dommett, G. H. B.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Graphene-Based Composite Materials. Nature 2006, 442, 282−286. (7) Gao, N.; Fang, X. Synthesis and Development of Graphene− Inorganic Semiconductor Nanocomposites. Chem. Rev. 2015, 115, 8294−8343. 28573
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces (29) Chang, H.; Tang, L.; Wang, Y.; Jiang, J.; Li, J. Graphene Fluorescence Resonance Energy Transfer Aptasensor for the Thrombin Detection. Anal. Chem. 2010, 82, 2341−2346. (30) Zheng, T.; Zhang, Q.; Feng, S.; Zhu, J.-J.; Wang, Q.; Wang, H. Robust Nonenzymatic Hybrid Nanoelectrocatalysts for Signal Amplification toward Ultrasensitive Electrochemical Cytosensing. J. Am. Chem. Soc. 2014, 136, 2288−2291. (31) Zhu, C.; Yang, G.; Li, H.; Du, D.; Lin, Y. Electrochemical Sensors and Biosensors Based on Nanomaterials and Nanostructures. Anal. Chem. 2015, 87, 230−249. (32) Dong, S.; Zhao, R.; Zhu, J.; Lu, X.; Li, Y.; Qiu, S.; Jia, L.; Jiao, X.; Song, S.; Fan, C.; Hao, R.; Song, H. Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza a (H7N9) Virus. ACS Appl. Mater. Interfaces 2015, 7, 8834−8842. (33) Tang, D.; Lin, Y.; Zhou, Q.; Lin, Y.; Li, P.; Niessner, R.; Knopp, D. Low-Cost and Highly Sensitive Immunosensing Platform for Aflatoxins Using One−Step Competitive Displacement Reaction Mode and Portable Glucometer−Based Detection. Anal. Chem. 2014, 86, 11451−11458. (34) Hou, L.; Zhu, C.; Wu, X.; Chen, G.; Tang, D. Bioresponsive Controlled Release from Mesoporous Silica Nanocontainers with Glucometer Readout. Chem. Commun. 2014, 50, 1441−1443. (35) Lu, X.; Wen, Z.; Li, J. Hydroxyl-Containing Antimony Oxide Bromide Nanorods Combined with Chitosan for Biosensors. Biomaterials 2006, 27, 5740−5747. (36) Shi, J.-J.; He, T.-T.; Jiang, F.; Abdel-Halim, E. S.; Zhu, J.-J. Ultrasensitive Multi-Analyte Electrochemical Immunoassay Based on Gnr-Modified Heated Screen-Printed Carbon Electrodes and PS@ PDA-Metal Labels for Rapid Detection of Mmp-9 and Il-6. Biosens. Bioelectron. 2014, 55, 51−56. (37) Zhang, X.; Chen, C.; Yin, J.; Han, Y.; Li, J.; Wang, E. Portable and Visual Electrochemical Sensor Based on the Bipolar Light Emitting Diode Electrode. Anal. Chem. 2015, 87, 4612−4616. (38) Xiang, Y.; Lu, Y. Using Personal Glucose Meters and Functional DNA Sensors to Quantify a Variety of Analytical Targets. Nat. Chem. 2011, 3, 697−703. (39) Yu, Z. G.; Lai, R. Y. Effect of Signaling Probe Conformation on Sensor Performance of a Displacement-Based Electrochemical DNA Sensor. Anal. Chem. 2013, 85, 3340−3346. (40) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J. Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor. Langmuir 2007, 23, 6827−6834. (41) Xuan, F.; Luo, X.; Hsing, I. M. Ultrasensitive Solution-Phase Electrochemical Molecular Beacon-Based DNA Detection with Signal Amplification by Exonuclease III-Assisted Target Recycling. Anal. Chem. 2012, 84, 5216−5220. (42) Luo, X.; Lee, T. M.-H.; Hsing, I. M. Immobilization-Free Sequence-Specific Electrochemical Detection of DNA Using Ferrocene-Labeled Peptide Nucleic Acid. Anal. Chem. 2008, 80, 7341−7346. (43) Xuan, F.; Fan, T. W.; Hsing, I. M. Electrochemical Interrogation of Kinetically-Controlled Dendritic DNA/PNA Assembly for Immobilization-Free and Enzyme-Free Nucleic Acids Sensing. ACS Nano 2015, 9, 5027−5033. (44) Wei, X.; Ma, X.; Sun, J. J.; Lin, Z.; Guo, L.; Qiu, B.; Chen, G. DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode. Anal. Chem. 2014, 86, 3563−3567. (45) Xuan, F.; Luo, X.; Hsing, I. M. Conformation-Dependent Exonuclease III Activity Mediated by Metal Ions Reshuffling on Thymine-Rich DNA Duplexes for an Ultrasensitive Electrochemical Method for Hg2+ Detection. Anal. Chem. 2013, 85, 4586−4593. (46) Xuan, F.; Luo, X.; Hsing, I. M. Sensitive Immobilization-Free Electrochemical DNA Sensor Based on Isothermal Circular Strand Displacement Polymerization Reaction. Biosens. Bioelectron. 2012, 35, 230−234. (47) Liu, S.; Lin, Y.; Wang, L.; Liu, T.; Cheng, C.; Wei, W.; Tang, B. Exonuclease III-Aided Autocatalytic DNA Biosensing Platform for
Immobilization-Free and Ultrasensitive Electrochemical Detection of Nucleic Acid and Protein. Anal. Chem. 2014, 86, 4008−4015. (48) Nie, J.; Zhang, D.-W.; Zhang, F.-T.; Yuan, F.; Zhou, Y.-L.; Zhang, X.-X. Reporter-Triggered Isothermal Exponential Amplification Strategy in Ultrasensitive Homogeneous Label-Free Electrochemical Nucleic Acid Biosensing. Chem. Commun. 2014, 50, 6211−6213. (49) Liu, B.; Zhang, B.; Chen, G.; Tang, D. An Omega-Like DNA Nanostructure Utilized for Small Molecule Introduction to Stimulate Formation of Dnazyme-Aptamer Conjugates. Chem. Commun. 2014, 50, 1900−1902. (50) Hu, C.; Yang, D.-P.; Wang, Z.; Yu, L.; Zhang, J.; Jia, N. Improved Eis Performance of an Electrochemical Cytosensor Using Three-Dimensional Architecture Au@BSA as Sensing Layer. Anal. Chem. 2013, 85, 5200−5206. (51) Sheng, Q.-L.; Liu, R.-X.; Zheng, J.-B.; Zhu, J.-J. Reversible Switches of DNA Nanostructures between ″Closed″ and ″Open″ States and Their Biosensing Applications. Nanoscale 2013, 5, 7505− 7511. (52) Li, X.; Shen, L.; Zhang, D.; Qi, H.; Gao, Q.; Ma, F.; Zhang, C. Electrochemical Impedance Spectroscopy for Study of Aptamer− Thrombin Interfacial Interactions. Biosens. Bioelectron. 2008, 23, 1624− 1630. (53) Cao, J.-T.; Hao, X.-Y.; Zhu, Y.-D.; Sun, K.; Zhu, J.-J. Microfluidic Platform for the Evaluation of Multi-Glycan Expressions on Living Cells Using Electrochemical Impedance Spectroscopy and Optical Microscope. Anal. Chem. 2012, 84, 6775−6782. (54) Akhavan, O.; Ghaderi, E.; Rahighi, R. Toward Single-DNA Electrochemical Biosensing by Graphene Nanowalls. ACS Nano 2012, 6, 2904−2916. (55) Akhavan, O.; Ghaderi, E.; Rahighi, R.; Abdolahad, M. Spongy Graphene Electrode in Electrochemical Detection of Leukemia at Single-Cell Levels. Carbon 2014, 79, 654−663. (56) Akhavan, O.; Ghaderi, E.; Hashemi, E.; Rahighi, R. UltraSensitive Detection of Leukemia by Graphene. Nanoscale 2014, 6, 14810−14819. (57) Tang, L.; Wang, Y.; Li, Y.; Feng, H.; Lu, J.; Li, J. Preparation, Structure, and Electrochemical Properties of Reduced Graphene Sheet Films. Adv. Funct. Mater. 2009, 19, 2782−2789. (58) Tang, D.; Zhang, B.; Tang, J.; Hou, L.; Chen, G. DisplacementType Quartz Crystal Microbalance Immunosensing Platform for Ultrasensitive Monitoring of Small Molecular Toxins. Anal. Chem. 2013, 85, 6958−6966. (59) Lin, Y.; Zhou, Q.; Lin, Y.; Tang, D.; Niessner, R.; Knopp, D. Enzymatic Hydrolysate-Induced Displacement Reaction with Multifunctional Silica Beads Doped with Horseradish Peroxidase−Thionine Conjugate for Ultrasensitive Electrochemical Immunoassay. Anal. Chem. 2015, 87, 8531−8540. (60) Li, F.; Chao, J.; Li, Z.; Xing, S.; Su, S.; Li, X.; Song, S.; Zuo, X.; Fan, C.; Liu, B.; Huang, W.; Wang, L.; Wang, L. Graphene OxideAssisted Nucleic Acids Assays Using Conjugated PolyelectrolytesBased Fluorescent Signal Transduction. Anal. Chem. 2015, 87, 3877− 3883. (61) Li, F.; Huang, Y.; Yang, Q.; Zhong, Z.; Li, D.; Wang, L.; Song, S.; Fan, C. A Graphene-Enhanced Molecular Beacon for Homogeneous DNA Detection. Nanoscale 2010, 2, 1021−1026. (62) Liu, X.; Aizen, R.; Freeman, R.; Yehezkeli, O.; Willner, I. Multiplexed Aptasensors and Amplified DNA Sensors Using Functionalized Graphene Oxide: Application for Logic Gate Operations. ACS Nano 2012, 6, 3553−3563. (63) Lu, C.-H.; Li, J.; Liu, J.-J.; Yang, H.-H.; Chen, X.; Chen, G.-N. Increasing the Sensitivity and Single-Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher. Chem. - Eur. J. 2010, 16, 4889−4894. (64) Yang, B.; Zhang, X.-B.; Kang, L.-P.; Shen, G.-L.; Yu, R.-Q.; Tan, W. Target-Triggered Cyclic Assembly of DNA−Protein Hybrid Nanowires for Dual-Amplified Fluorescence Anisotropy Assay of Small Molecules. Anal. Chem. 2013, 85, 11518−11523. (65) Zhao, X.-H.; Gong, L.; Zhang, X.-B.; Yang, B.; Fu, T.; Hu, R.; Tan, W.; Yu, R. Versatile Dnazyme-Based Amplified Biosensing 28574
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
Research Article
ACS Applied Materials & Interfaces Platforms for Nucleic Acid, Protein, and Enzyme Activity Detection. Anal. Chem. 2013, 85, 3614−3620. (66) Wu, D.; Xin, X.; Pang, X.; Pietraszkiewicz, M.; Hozyst, R.; Sun, X. G.; Wei, Q. Application of Europium Multiwalled Carbon Nanotubes as Novel Luminophores in an Electrochemiluminescent Aptasensor for Thrombin Using Multiple Amplification Strategies. ACS Appl. Mater. Interfaces 2015, 7, 12663−12670. (67) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339−1339. (68) Brooksby, P. A.; Downard, A. J. Electrochemical and Atomic Force Microscopy Study of Carbon Surface Modification Via Diazonium Reduction in Aqueous and Acetonitrile Solutions. Langmuir 2004, 20, 5038−5045. (69) Wang, Z.; Zhou, X.; Zhang, J.; Boey, F.; Zhang, H. Direct Electrochemical Reduction of Single-Layer Graphene Oxide and Subsequent Functionalization with Glucose Oxidase. J. Phys. Chem. C 2009, 113, 14071−14075. (70) Dikin, D. A.; Stankovich, S.; Zimney, E. J.; Piner, R. D.; Dommett, G. H. B.; Evmenenko, G.; Nguyen, S. T.; Ruoff, R. S. Preparation and Characterization of Graphene Oxide Paper. Nature 2007, 448, 457−460. (71) Park, S.; Ruoff, R. S. Chemical Methods for the Production of Graphenes. Nat. Nanotechnol. 2009, 4, 217−224. (72) Tang, Z.; Mallikaratchy, P.; Yang, R.; Kim, Y.; Zhu, Z.; Wang, H.; Tan, W. Aptamer Switch Probe Based on Intramolecular Displacement. J. Am. Chem. Soc. 2008, 130, 11268−11269.
28575
DOI: 10.1021/acsami.5b09932 ACS Appl. Mater. Interfaces 2015, 7, 28566−28575
