Direct and Freely Switchable Detection of Target Genes Engineered

Dec 20, 2012 - Unlike most electrochemical preparation of hybrids based on rGNO and polymer, electrochemical synthesis of PABSA (during the pulse ...
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Direct and Freely Switchable Detection of Target Genes Engineered by Reduced Graphene Oxide-Poly(m‑Aminobenzenesulfonic Acid) Nanocomposite via Synchronous Pulse Electrosynthesis Tao Yang, Qian Guan, Xiuhong Guo, Le Meng, Meng Du, and Kui Jiao* State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China S Supporting Information *

ABSTRACT: A novel one-step electrochemical synthesis of the reduced graphene oxide and poly(m-aminobenzenesulfonic acid, ABSA) nanocomposite (PABSA−rGNO) via pulse potentiostatic method (PPM) for direct and freely switchable detection of target genes is presented. Unlike most electrochemical preparation of hybrids based on rGNO and polymer, electrochemical synthesis of PABSA (during the pulse electropolymerization period of PPM) and electrochemical reduction of rGNO (during the resting period of PPM), in this paper, were alternately performed. The total progress synchronously resulted in PABSA−rGNO nanocomposite. This nanocomposite was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier Transform infrared spectroscopy (FT-IR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The PABSA−rGNO nanocomposite integrated graphene (a single-atom thick, two-dimensional sheet of sp2 bonded conjugated carbon) with PABSA (owning rich-conjugated structures, functional groups, and excellent electrochemical activity), which could serve as an ideal electrode material for biosensing and electrochemical cell, etc. As an example, the immobilization of the specific probe DNA was successfully conducted via the noncovalent method due to the π−π* interaction between conjugated nanocomposite and DNA bases. The hybridization between the probe DNA and target DNA induced the product dsDNA to be released from conjugated nanocomposite, accompanied with the self-signal regeneration of nanocomposite (“signal-on”). The self-signal changes served as a powerful tool for direct and freely switchable detection of different target genes, and the synergistic effect of PABSA−rGNO nanocomposite effectively improved the sensitivity for the target DNA detection.

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graphene composites has emerged as a green and fast approach with in situ precise control.22,23 A polyaniline (PANI)/ graphene composite paper was fabricated by an in situ anodic electropolymerization of polyaniline film on graphene.22 Ma’s group used graphite oxide and aniline monomer as the starting materials to synthesize PANI/graphene composite film by a facile one-step electrodeposition method,23 where the graphene oxide was electrochemically reduced by cyclic voltammetry. On the basis of the outstanding property of the composite film, a H2O2 biosensor and a supercapacitor had been constructed. Compared with the pristine polyaniline, one of sulfonated polyanilines, poly(aminobenzenesulfonic acid), possesses excellent electrochemical activities24 and electrocatalytic ability in neutral solutions.25 However, the electrochemical activity of pristine polyaniline was limited in acidic buffer.26,27 Poly(aminobenzenesulfonic acid) has been integrated with many materials to fabricate nanocomposites including carbon nanotubes,28−30 polypyrrole,31 PANI,32 and poly(amido amine).33 For example, sulfonated polyaniline (SPANI) has been

mong the reduction methods of graphene oxide, electrochemical reduction has been the focus for the benefits of being simple, green, without toxic agent addition, etc.1−3 However, the most adopted chemical methods usually use hydroquinone, NaBH4, hydrazine hydrate, hydrazine vapor, or hydrazine with NH3 as reducing agent.4,5 The electrochemical reduced graphene oxide (ERGNO) has usually been obtained by two steps: (1) drop-casting of graphene oxide (obtained from ultrasound stripping of graphite oxide (GO)) solution on basal electrodes; (2) then, electrochemical reduction of above modified electrodes.6 A series of electrochemical methods, such as cyclic voltammetry (CV)7−12 and potentiostatic method,13−18 have been employed. However, the pulse potentiostatic method (PPM),19,20 having advantages, including simplicity, time savings, low temperature growth without any heat treatment, and high purity of the deposits, has not been adopted in the ERGNO field. Graphene-based hybrids have attracted great interest in the science and technology fields. In these fields, graphene is regarded as an ideal platform for designing composite nanomaterials serving as high-performance electrocatalytic or electrochemical devices, etc.21 Especially, an in situ electrochemical method constructing the conducting polymer and © 2012 American Chemical Society

Received: July 13, 2012 Accepted: December 20, 2012 Published: December 20, 2012 1358

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ability and the water resistance34 of the reduced graphene oxide−poly(m-aminobenzenesulfonic acid) nanocomposite (PABSA−rGNO). The PABSA−rGNO presents excellent electrochemical activity and thermal stability even in neutral environment. The immobilization of the probe DNA was successfully conducted via the noncovalent method due to π−π* interaction between conjugated nanocomposite and DNA bases, resulting in electrochemical self-signals decrease. However, when the probe modified eletrode hybridized with target DNA sequences, the resulted dsDNA was released from conjugated nanocomposite, accompanied with the regeneration of self-signals. The synergistic effect of graphene-based nanocomposite and the application of sensitive EIS detection method improved the sensitivity for the target DNA detection. The obtained PABSA−rGNO by PPM will be helpful for extending the application of graphene-based nanocomposite in the electrochemical capacitors and biosensing field.

successfully used in the noncovalent functionalization of graphene. Due to the existence of sulfonated polyaniline, the composite exhibited high conductivity, good electrocatalytic activity, and stability.34 The dropcast film of SPANI-modified graphene is stable in aqueous media being ascribed to the strong interactions between SPANI chains and the basal planes of graphene. In DNA nanobiosensors, immobilization of probe DNA on sensing platform plays a key role, which directly determined the sensitivity of assay. In recent years, graphene or organic conjugated molecules, having rich-conjugated structures, functional groups, and excellent electrochemical activity, have emerged to efficiently immobilize the probe DNA via π−π* interaction between conjugated interface and DNA bases. It should be noted that, after hybridization, the resulted dsDNA will be released from conjugated interface and induce the signal changes of nanointerface (called “signal-on” or “signal-off”). The signal changes could serve as a powerful tool for freely switchable detection of the different gene sequences.35−38 At the same time, direct and reagentless DNA electrochemical detection39 seems to be very eye-catching due to device miniaturization, low cost, simplicity, and no outer indicators or complicated label.40,41 To the best of our knowledge, graphene−organic conjugated molecules nanocomposite has not been reported for direct, highly sensitive, and freely switchable detection of target genes. At the same time, graphene and its nanocomposites have been explored to fabricate various fascinating electrochemical DNA biosensors for the direct electrochemical oxidation of DNA bases and DNA hybridization sensing.42 In the former case, some works have demonstrated that graphene possesses excellent electrochemical catalytic activity to direct electrooxidation behaviors of adenine and guanine.2,43−45 In the latter case, the immobilization of DNA on graphene or its nanocomposites is mainly conducted via covalent grafting, self-assembly, or electrostatic adsorption. The detection of the target gene depended on two routes: (1) using the redox intercalaters (e.g., methylene blue,46 daunomycin47,48 etc.) with voltammetric detection; (2) using [Fe(CN)6]3−/4−, a classic redox probe, with electrochemical impedance spectroscopic (EIS) detection. For example, Niu’s group prepared PTCA/ graphene/GCE (3,4,9,10-perylene tetracarboxylicacid, PTCA),49 Au-IL/PTCA/graphene/GCE (ionic liquid, IL),50 and PDI/graphene/GCE (N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide, PDI)51 via covalent grafting or electrostatic interaction to immobilize DNA probe. EIS can monitor the immobilization and hybridization of DNA by adopting [Fe(CN)6]3−/4− as redox probe. The approach did not need labeled oligonucleotide probes or targets, showing advantage in terms of simplicity and noninvasiveness. Thus, it is worth mentioning that the noncovalent π−π* stacking, which was widely used in the field of optical DNA biosensors,52−54 has been applied in electrochemical DNA biosensors.9,55 Pumera’s group employed π-stacking interactions between the ring of nucleobases and the hexagonal cells of graphene to immobilize DNA probe.35 In this paper, the reduced graphene oxide (rGNO) and the poly(m-aminobenzenesulfonic acid), PABSA, were synchronously obtained via a facile and effective PPM. The π−π* interaction and hydrogen bonding between the GNO layers and aromatic rings monomer (m-aminobenzenesulfonic acid, ABSA) promote efficient polymerization, the film-forming

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EXPERIMENTAL SECTION Apparatus. PPM and electrochemical measurements were conducted with a CHI 660D electrochemical workstation (Shanghai CH Instrument Company, China) and a standard three-electrode cell containing a home-made carbon paste modified working electrode, a saturated calomel reference electrode (SCE), and a platinum wire auxiliary electrode. The resulted composite was characterized by scanning electron microscopy (SEM) (JSM−6700F machine (JEOL, Tokyo, Japan)), transmission electron microscopy (TEM) micrographs (JEM 2100 transmission electron microscopy), Fourier Transform infrared spectroscopy (FT-IR) spectrum (Tensor 27 FTIR spectrophotometer (Bruker Company, Germany)), and X− ray powder diffraction (XRD) (Bruker D8 advanced X-ray diffractometer using Cu Kα radiation, operated at 40 kV and 30 mA). Reagents. ABSA (purity >98.0%) was purchased from Fluka (USA). Natural graphite (spectral pure, about 30 μm) was obtained from Sinopharm Chemical Reagent Co., Ltd. Tris (hydroxymethyl) amminomethane (Tris) was acquired from Sigma (St. Louis, MO, USA). Sodium dodecyl sulfonate (SDS) was purchased from Shanghai Reagent Company and used as received. All the chemicals were of analytical grade. All aqueous solutions were prepared with ultrapure water from an Aquapro ultrapure water system (Ever Young Enterprises Development. Co. Ltd., Chongqing, China). The 18-base synthetic oligonucleotides probe (pDNA), its complementary DNA (cDNA, target DNA, namely, an 18-base fragment of PML/RARA fusion gene sequence formed from promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA)), single-base mismatched DNA, and noncomplementary sequence DNA (ncDNA) were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Their base sequences and stock solutions were the same as those in ref 56. Similarly, the PAT gene sequences were the same as those in ref 57. Preparation and Modification of Electrodes. The fabrication of carbon paste electrode (CPE) was carried out using the method reported by Yang.57 Graphene oxide (GNO) was synthesized according to our previous protocol.2 Ten microliters of the mixed liquor (10.0 mg of GNO and 1.0 mL of 0.04 mol/L ABSA dispersed in 100 mL of 1.0 mol/L H2SO4) was dripped on the fresh CPE surface and naturally dried in the air.34 After that, the modified electrode was immersed in ABSA-free 1.0 mol/L H2SO4 solution58 ready for 1359

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Figure 1. SEM and TEM images of the GNO (A, B), rGNO(C, D), PABSA (E, F), and PABSA−rGNO (G, H).

tion, only a few of the ABSA monomers existing in the network were consumed. Accompanied with the alternate pulse electropolymerization section and resting section, electropolymerization continued, until the ABSA monomer existing in the microcompartment was completely consumed. The

electropolymerization by PPM. The electrolyte solution can wet the interface and permeate into59 the open and loose ABSA-GNO/CPE (Supporting Information, Figure S1). Thus, a microcompartment containing ABSA monomer and supporting solution is formed. At the beginning of electropolymeriza1360

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Figure 2. Schematic of the electropolymerization of ABSA adopting the potentiostatic method (section b) and the PPM (section c).

veil waves is intrinsic to graphene nanosheets.13 After the electropolymerization of the ABSA monomers by PPM without GNO, the formed PABSA displays a uniform and smooth surface,60 shown in Figure 1E,F. In Figure 1G,H, the resulted PABSA−rGNO nanocomposite reveals plenty of particle-like protuberances61 compared with the rGNO film and PABSA film. The formed homologous structure of the composite film maybe originated from the GNO which served as a support material and offered a large number of active sites for nucleation of ABSA initially.62,63 Meanwhile, accompanied with the synchronal reduction of the GNO, the electropolymerization of PABSA will prefer to grow along with the platform of rGNO due to the self-assembly effect between the conjugated rGNO and the conjugated ABSA.23 In addition, it is hard to observe individual rGNO or PABSA polymer, which indicates the PABSA and rGNO had been integrated successfully. It can be seen that the prepared PABSA−rGNO nanocomposite has a large specific area, which is helpful for the bio-immobilization. The schematic image shown in Figure 2 illustrates the whole pulse electropolymerization process. In the pulse electropolymerization section (Figure 2 section a), the ABSA monomers in the immediate vicinity of the graphene oxide film had been consumed, accompanied with emergence of electropolymerized PABSA nanoparticles on the graphene surface. If the electropolymerization process is performed continuously (keeping potential at 2.2 V stable like the potentiostatic method, Figure 2 section b), the process of polymerization is uninterrupted, and the initially resulted PABSA nanoparticles on the surface would serve as seeds or nucleation sites for the followed ABSA monomers, which are ready for electropolymerization in the intercalating spaces between graphene oxide sheets in the microcompartment. Due to the electropolymerization being inclined to continue on any given polymer chain rather than nucleate growth of a new chain,20 larger sized PABSA nanoparticles appear. Finally, all of the ABSA monomers in the microcompartment were consumed, and a uniform PABSA film is coated on the graphene oxide.62 However, if the potential shifted to negative direction (Figure 2, resting time, section c) after an unremitting anodic deposition for a given time, the graphene oxide was only partly

obtained electrode was denoted as PABSA−rGNO/CPE. The optimal parameters of electropolymerization were listed as follows: upper limit potential Ea, 2.2 V; lower limit potential Ec, −1.5 V; anodic pulse duration Ta, 0.7 s; cathodic pulse duration Tc, 0.3 s; total pulse time Texp, 400 s. PABSA/CPE and rGNO/ CPE were prepared under the same procedure as PABSA− rGNO/CPE preparation just without GNO or ABSA existing, respectively. Immobilization and Hybridization of pDNA on PABSA−rGNO. A 10.0 μL Tris−HCl buffer solution (pH 7.0) containing 1.0 × 10−6 mol/L pDNA was pipetted onto the PABSA−rGNO/CPE, air-dried to dryness, and then rinsed with ultrapure water to remove the unimmobilized oligonucleotides. Afterward, 10.0 μL of hybridization solution (2× SSC buffer, pH 7.0) containing cDNA was dropped onto the probemodified electrode to perform the hybridization reaction. To remove the unbonded oligonucleotides, the electrode was thoroughly washed with 0.2% SDS solution. The same procedures as mentioned above were applied to the probemodified electrodes for hybridization with single-base mismatched and noncomplementary sequences. Electrochemical Measurements. The following parameters were employed for CV: scan rate, 100 mV/s; potential scanning range from 0.6 to −0.3 V. Supporting electrolyte was 0.30 mol/L PBS solution (pH 7.0) or 2.0 mmol/L K3Fe(CN)6 and 2.0 mmol/L K4Fe(CN)6 (1:1) solution containing 0.1 mol/L KCl. The EIS was measured from 0.01 Hz to 105 Hz with AC voltage amplitude of 5 mV. The supporting electrolyte is the same as that adopted in the CV measurement. The EIS measurements were carried out under open-circuit condition. The reported result for every electrode in this assay was the mean value of three parallel measurements.

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RESULTS AND DISCUSSION Characterization and Mechanism of the PABSA− rGNO. SEM and TEM images of the GNO, rGNO, PABSA, and PABSA−rGNO are shown in Figure 1. GNO film is smooth with a little wrinkled paper-like structure (Figure 1A, B). Compared with GNO, the rGNO (prepared by PPM with only GNO existing) is corrugated and scrolled after the reduction (Figure 1C, D). The resemblance of crumpled silk 1361

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spacing of 0.342 nm in the rGNO sample reveals a broad reflection peak centering around 26.06°.64 Two peaks were observed centered at 2θ = 21.32° and 23.74°, being related to the d-spacing of PABSA−rGNO (0.416 nm) and rGNO/ rGNO (0.374 nm), respectively.34 The diffraction peaks at 29.74° (0 2 0), 35.78° (1 2 0), and 38.7° (1 2 1) were ascribed to the existence of the highly crystalline phase of PABSA.67 Therefore, the XRD result further confirms that PABSA film has been coated on the rGNO nanosheets, which is also consistent with the results of FT-IR investigation. Electrochemistry of the PABSA−rGNO. The electrochemical behaviors of CPE (a), GNO/CPE (b), rGNO/CPE (c), PABSA/CPE(d), and PABSA−rGNO/CPE (e) were checked in 2.0 mmol/L [Fe(CN)6]3−/4− containing 0.10 mol/ L KCl with CV from 0.6 to −0.3 V (shown in Figure 4A). For

electrochemically reduced, and at the same time, the diffusion of the ABSA monomers between the intercalating spaces of GNO sheets appeared. The short resting period not only prompts the electrochemical reduction of GNO but also benefits the next pulse deposition for ABSA electropolymerization. The process (without shift of positive potential) could let ABSA monomers look for new nucleation sites, such as the oxidized defects of intercalating spaced GNO, rather than directly being deposited on initially resulted PABSA nanoparticles in the electropolymerization pulse just as discussed above. The combined effect is the reason why the polymer on the graphene surface takes the form of plenty of particle-like protuberances (Figure 2, lower inset, SEM (C)) rather than large smooth sheets (Figure 2, upper inset, TEM (A), SEM (B)). FT-IR and XRD of the PABSA−rGNO. The existence of the functional groups on the PABSA−rGNO nanocomposite can be confirmed by the FT-IR results (shown in Figure 3A).

Figure 3. FT-IR spectra (A) and XRD pattern (B) of PABSA−rGNO.

The peak at 1635 cm−1 is attributed to aromatic CC as well as bands at 1399 cm−1 to carboxy C−O and 1382 cm−1 to alkoxy C−O groups located at the edges of the GNO nanosheets.64 The peak at 1227 cm−1 ascribes to epoxy C−O groups. The shoulders at 2849 and 2918 cm−1 correspond to aromatic sp2 C−H stretching of graphene.65 The main peaks at 1585 and 1463 cm−1 can be assigned to the stretching vibrations of benzene rings. The peak at 1228 cm−1 is attributed to the C−N stretching vibration.23 The bands at 1059, 719, and 595 cm−1 can be assigned to the OSO stretching vibration, S−O stretching vibration, and C−S stretching vibration, respectively.66 Meanwhile, a broad band at ca. 3431 cm−1 can be seen due to the overlapping of the strong absorption of the N−H stretching vibrations of PABSA and that of OH groups of graphene.65 On the basis of all of the above peaks, it can be seen that PABSA−rGNO nanocomposite has integrated PABSA and GNO effectively. The structure of the nanocomposite was also investigated by XRD measurements, which is shown in Figure 3B. An interlayer

Figure 4. Representative CVs (A) and Nyquist diagrams (B) of 2.0 mmol/L [Fe(CN)6]3−/4− (1:1) containing 0.1 mol/L KCl; CVs (C) in 0.30 mol/L PBS (pH 7.0) recorded at bare CPE (a), GNO/CPE (b), rGNO/CPE (c), PABSA/CPE (d), and PABSA−rGNO/CPE (e).

PABSA−rGNO/CPE, the redox peak currents increase dramatically in comparison with the other four electrodes, and the ΔEp shrinks very obviously (148 mV). The results demonstrated that the high specific surface area and electrical conductivity of the PABSA−rGNO facilitated the electron transfer of [Fe(CN)6]3−/4−. The related active surface areas (average of three measurements) of rGNO/CPE (0.275 ± 0.007 cm2), PABSA/CPE (0.289 ± 0.008 cm2), and PABSA− rGNO/CPE (0.448 ± 0.003 cm2) were estimated.68 Herein, the synergistic effect of PABSA and rGNO on enhancing the 1362

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electroactive surface area is outstanding. For details, please see the Supporting Information. Similarly, in comparison with the other four modified electrodes (Ret values of CPE, GNO/CPE, rGNO/CPE, and PABSA/CPE estimated to be 23 400, 34 900, 960, and 730 Ω, respectively), the lowest Ret value (320 Ω) in EIS occurs after the PABSA−rGNO forms (Figure 4B, curve e), which is consistent with the above CV results using [Fe(CN)6]3−/4− as electrochemical probe. Note: Ret, electron transfer resistance.57 Figure 4C shows the CVs of the electrodes (corresponding to Figure 4A) recorded in 0.30 mol/L PBS (pH 7.0). PABSA− rGNO/CPE (Figure 4C, curve e) shows well-defined redox peaks at ca. 0.103 and 0.073 V, respectively, indicating redox responses of PABSA in a neutral pH environment.60 All results above indicated that the existence of rGNO enhanced the redox behaviors of the PABSA. Here, it should be noted that sulfonated polyaniline modified electrode was normally unstable, which is easy to escape from the electrode surface. However, sulfonated polyaniline films fabricated by electrochemical copolymerization69 or by layerby-layer deposition32,33 tend to be stable. Just as Shi’s group stated,34 the π−π* interaction and hydrogen bonding between the graphene oxide layers and aromatic rings monomer (ABSA) greatly improve the film-forming ability and the water resistance of the PABSA−rGNO nanocomposite, which is stable in the neutral environment. Optimization of the Fabrication of the PABSA−rGNO. The PPM includes several parameters, Ea, Ec, Ta, Tc, and Texp,60 where Ea and Ec are the upper limit potential and lower limit potential in PPM, respectively, and Ta, Tc, and Texp represent the anodic pulse duration, cathodic pulse duration, and total pulse time, respectively. The five important electropolymerization parameters are prominently crucial to the formation of PABSA−rGNO. The conductivity of the PABSA−rGNO was affected by the values of Ea, Ec, and it’s related polymerization time, Texp. For the details of the optimization, see the Supporting Information. Immobilization and Hybridization of DNA. After the immobilization and hybridization of DNA on the PABSA− rGNO layer, the CV and EIS techniques were applied to investigate the self-redox property changes of the PABSA− rGNO layer in 0.30 mol/L PBS (pH 7.0). The results are shown in Figure 5. When the probe DNA (pDNA) was noncovalently assembled on the PABSA−rGNO layer through π−π* stacking,35,36 the redox peaks of PABSA−rGNO could be hardly viewed (Figure 5A, curve a). This is ascribed to the π−π* stacking interaction between the ring of nucleobases and the rich-conjugated structures of the nanocomposite, as reported previously.35,36 The ssDNA coated on the surface of the film blocked the effective electron transfer channel of the PABSA−rGNO. Besides, when the DNA probe film was coated on the electrode surface, the steric hindrance was introduced at the same time. Therefore, the response of the PABSA−rGNO layer shrinks (“signal-off”). In comparison with Figure 4C curve e, the obvious decrease of CV current could prove that the pDNA had been immobilized on the PABSA−rGNO layer successfully. After hybridization with the complementary DNA (cDNA), a significant increase of CV currents (Figure 5A, curve b) is observed in comparison with curve a. A couple of redox peaks appears again. The stronger and more stable binding between pDNA and cDNA, compared with π−π* stacking previously, will induce the resulting dsDNA helix to be released from the surface of conjugated nanocomposite, accompanied

Figure 5. (A) Representative CVs of the PABSA−rGNO/CPE after pDNA immobilization (a), hybridization reaction (hybridized with 1.0 × 10 −10 mol/L cDNA, b) recorded in 0.30 mol/L PBS (pH 7.0); (B) representative Bode plots recorded at PABSA−rGNO/CPE (a), pDNA/PABSA−rGNO/CPE (b), dsDNA/PABSA−rGNO/CPE (hybridized with cDNA) (c), hybridized with single-base mismatched DNA (d), and the probe electrode hybridized with ncDNA (e) in 0.30 mol/L PBS (pH 7.0) under open-circuit conditions.

with the self-signal regeneration of nanocomposite (“signalon”). EIS is often adopted in the biorecognition events taking place between the surface and the electrolyte interface, which was also applied to directly detect the DNA immobilization and hybridization without adding any electroactive indicator.70−74 Herein, the related Bode plots are shown in Figure 5B. Curve a is the Bode plot of PABSA−rGNO/CPE. The impedimetric value increases obviously when the pDNA was adsorbed onto PABSA−rGNO nanosheets (curve b) due to π−π* stacking, indicating the successful immobilization of the pDNA. The result is consistent with the above CV measurements. After the pDNA hybridized with the complementary DNA sequence (curve c), the impedimetric value decreases. The escape of dsDNA from the surface of conjugated nanocomposite induces “signal-on”. The single-base mismatched sequence (curve d) and noncomplementary sequence (ncDNA, curve e) are also shown in Figure 5B. The value of the impedance related with single-base mismatched sequences was higher than the curve c, illustrating that part hybridization happened. However, when the pDNA/PABSA−rGNO/CPE was hybridized with ncDNA, the impedimetric value hardly varies. These results showed that this indicator-free impedance biosensing of DNA hybridization based on PABSA−rGNO nanocomposite exhibited good selectivity. Detection of PML/RARA Fusion Gene Sequence. The pDNA/PABSA−rGNO film was used to detect different concentrations of the PML/RARA fusion gene sequence.56 The obtained Bode plots in 0.30 mol/L PBS (pH 7.0) are shown in Figure 6A. The difference of the log Z value (namely, Δlog Z) at 103 Hz between the probe-captured electrode and that after hybridization with cDNA was adopted as the direct measurement signal.71 The Δlog Z value was linear with the 1363

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outer indicators35,76 or complex labels to indicate the immobilization and hybridization.77−79 In the DNA sensors, the stability of oligonucleotide probes on the sensing surface is believed to be a key factor to achieve practical application. With the aim at investigating the stability of pDNA/PABSA−rGNO/CPE, the electrode was incubated in ultrapure water, Tris−HCl solution (pH 7.0), 2× SSC solution (pH 7.0), and PBS buffer solution (pH 7.0) at 25 °C for 6 h, respectively, and then tested in 0.30 mol/L PBS (pH 7.0). The EIS results showed that the incubated electrode kept the similar electrochemical activity compared with the original electrode and could be applied to the further quantitative assay of complementary DNA sequence. In addition, the pDNA/ PABSA−rGNO/CPE was stored at 4 °C for 10 days, and the impedance response remained 92.5%. Through the above tests, the results demonstrated that the prepared DNA biosensor revealed good stability. Besides, the reproducibility of the DNA biosensor is also highly significant in the DNA assay. The six independently prepared pDNA/PABSA−rGNO/CPEs revealed a relative standard deviation (R.S.D.) of 4.53% for the Δlog Z value at 103 Hz (before and after hybridization, just as illustrated in Figure 5B, curve b to c),56 showing the good reproducibility of the electrochemical DNA biosensor.

Figure 6. (A) Representative Bode plots of pDNA/PABSA−rGNO/ CPE (a) and after being hybridized with its complementary PML/ RARA gene sequence of different concentrations: 1.0 × 10−16 mol/L (b) to 1.0 × 10−8 mol/L (j). Conditions were the same as in Figure 5B. (B) The plot of Δlog Z vs the logarithm of target sequence concentrations. Each point is the mean of three measurements, and the error bars correspond to the standard deviation.

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logarithm of the concentrations of PML/RARA gene target sequence (Figure 6B). The dynamic detection range for the sequence-specific DNA of target gene was from 1.0 × 10−16 mol/L to 1.0 × 10−8 mol/L with the regression equation Δlog Z = 0.0294 log C + 0.4861, and the regression coefficient (γ) is 0.9889. With the adoption of highly sensitive EIS technology and the high electrochemical activity of PABSA−rGNO, the constructed direct and “signal-on” electrochemical DNA sensor could recognize the target gene sequence with a low detection limit of 3.7 × 10−17 mol/L. Switchable Ability, Stability, and Reproducibility of the Biosensor. To show the switchable ability of our constructed DNA sensing platform, as an example, the PAT gene (the related sequences illustrated in ref 57) was also detected through PABSA−rGNO film via CV (Figure 7). A

CONCLUSION In summary, we have reported a green, rapid, and simple method to prepare the PABSA−rGNO nanocomposite using a PPM technique, where the reduction of graphene oxide and the electropolymerazition of ABSA were synchronously performed. The nanocomposite integrated the advantages of the rGNO and PABSA, having rich-conjugated structures, good conductivity, functional groups, and biocompatibility. In virtue of the π−π* interaction between conjugated nanocomposite and conjugated molecules, the PABSA−rGNO could serve as favorable sensing platform for biomolecules immobilization, such as DNA. The excellent self-redox response could sensitively monitor the changes induced by hybridization of the target sequences. Especially, due to the release of the resulting dsDNA from PABSA−rGNO inducing the regeneration of the sensing platform, the freely switchable detection of different targets could be easily achieved. Our constructed nanocomposite could offer lots of opportunities for other biomolecules or electrochemical capacitors.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 7. Representative CVs of the PABSA−rGNO/CPE after PAT gene immobilization (a) and hybridization reaction (b) recorded in 0.30 mol/L PBS (pH 7.0).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-532-84022665. Fax: +86-532-84023927. Notes

similar tendency could be observed for the successful immobilization induced “signal-off” (Figure 7, curve a) and the ideal hybridization “signal-on” (Figure 7, curve b). From these results, the prepared DNA sensing platform could freely detect the different target genes just like the optical sensors.36,75 It should be noted that the signal of our switchable DNA sensor is directly relying on the redox responses of the functional nanocomposite, unlike most switchable DNA sensors with

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21275084, 20975057, 20805025), Doctoral Foundation of the Ministry of Education of China (No. 20113719130001), Outstanding Adult-Young Scientific 1364

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Research Encouraging Foundation of Shandong Province (No. BS2012CL013), and Scientific and Technical Development Project of Qingdao (No. 12-1-4-3-(23)-jch).

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