Chemically Modified Graphene and Sulfonic Acid-Doped Polyaniline

Apr 9, 2015 - Han Wang,. †. Tong Ge,. ‡. Tao Yang,*. ,†. Shizhong Luo,. † and Kui Jiao. †. †. Key Laboratory of Sensor Analysis of Tumor M...
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Chemically Modified Graphene and Sulfonic Acid-Doped Polyaniline Nanofiber Composites: Preparation Routes, Characterization, and Comparison of Direct DNA Detection Xinxing Wang,† Han Wang,† Tong Ge,‡ Tao Yang,*,† Shizhong Luo,† and Kui Jiao† †

Key Laboratory of Sensor Analysis of Tumor Marker of Education Ministry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China ‡ Huangdao Entry-Exit Inspection and Quarantine Bureau, Qingdao 266555, P. R. China S Supporting Information *

ABSTRACT: Recently, functional composites based on chemically modified graphenes (CMGs) and nanostructured conducting polymers have attracted wide interest in the field of electrochemical biosensing. However, comprehensive studies of the effects of various CMGs on the electrochemical properties and biosensing performance of the resulting composites are scarce. In this work, for the first time, we fabricated and deeply evaluated three composites composed of CMGs and sulfonic acid-doped polyaniline nanofiber (namely, CMG−SPAN composites). The CMGs (involving the unreduced form and reduced forms prepared by different reduction routes) were chosen to show the effects of reduction and different preparation routes on the morphologies, electrochemical properties, and DNA biosensing performances of the composites. Notably, the selfredox signals of SPAN in these composites were significantly enhanced and were used for rapid, direct, and label-free DNA detection. Moreover, a preliminary study of the capacitive characteristics of the thermally reduced graphene oxide−SPAN composite was conducted at the end of this work, owing to the potential benefits of the composite in a supercapacitor that were surprisingly observed in this research. The findings of this work will provide useful guides for better understanding of the interaction between CMG and SPAN and for the future development of high-performance functional materials for electrochemical sensors/biosensors and supercapacitors.

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from the ultrathin CMG sheets and their high conductivities contributed by the conducting polymer nanostructures.6 Similarly to pristine graphene, CMGs7 and CMG-based composites8,9 are also electrode materials that are being widely investigated and utilized in electrochemical sensing and biosensing research. CMGs of different types exhibit different oxygen functionalities, structures, and amounts of intrinsic defects, leading to different electrochemical behaviors.10,11 Several comparative studies on the electrochemical sensing and biosensing performances of these CMGs were conducted by Pumera’s group.12−14 They found that the differences in surface oxygen functionalities, structures, and defects of CMGs have a significant effects on their electrochemical behaviors in detecting the oxidation of DNA bases and DNA hybridization and on their electrocatalytic activities toward the oxidation of NADH, acetaminophen, and hydroquinone. However, compared to that on CMGs, the research on CMG-based composites is insufficient. For example, the effects of various CMGs on the electrochemical properties and

raphene has attracted much research attention in recent years and has shown great promise in many applications, such as electronics, energy storage/conversion, and bioscience/ biotechnologies.1 In particular, it has been widely studied as a promising type of electrode material in the fields of electrochemical sensing and biosensing, because of its high electrical conductivity, large surface area, and high heterogeneous electron-transfer rate.2 In view of the fact that pristine graphene is hard to produce on a large scale, chemically modified graphenes (CMGs) such as graphene oxide (GNO) and various reduced graphene oxides (rGNOs) are usually used as substitutes. These CMGs not only can be produced in large quantities by simple and low-cost methods, but also are more suitable for synthesizing highperformance CMG-based composites, because of the multiple functional groups decorating the surfaces of the CMG layers.3,4 To date, various CMG-based composites prepared by incorporating CMGs with one or more other components such as polymers, inorganic nanostructures, and carbon nanofillers have been fabricated. The combination of CMGs with other components brings enhanced performances and even synergistic effects that promote the wide application of these composite materials.5 Among these materials, composites composed of nanostructured conducting polymer are of special interest, owing to their large specific surface areas inherited © 2015 American Chemical Society

Received: January 18, 2015 Revised: April 8, 2015 Published: April 9, 2015 9076

DOI: 10.1021/acs.jpcc.5b00534 J. Phys. Chem. C 2015, 119, 9076−9084

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The Journal of Physical Chemistry C Scheme 1. Preparation Routes of the GNO−SPAN/CPE, erGNO−SPAN/CPE, and trGNO−SPAN/CPE

were collected using a model pHS-25 digital acidimeter (Shanghai Leici Factory, Shanghai, China). The morphologies of the resulting composites were characterized by scanning electron microscopy (SEM; JSM-6700F, JEOL, Tokyo, Japan). Carbon powder and paraffin were purchased from Shanghai Colloid Laboratory and Shanghai Hua Ling Healing Appliance Factory, respectively. Natural graphite (spectrally pure, ∼30 μm) was obtained from Sinopharm Chemical Reagent Co., Ltd. Tris(hydroxymethyl)amminomethane (Tris) was acquired from Sigma (St. Louis, MO). Sodium dodecyl sulfate (SDS) was purchased from Shanghai Reagent Company (Shanghai, China) and used as received. The trGNO was provided by Shanghai Second Polytechnic University. All chemicals used in this study were of at least analytical grade, and the 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 oligonucleotide probe (pDNA), its complementary DNA [cDNA; target DNA, namely, an 18-base fragment of PML/RARA fusion gene sequence generated from promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA)], single-base mismatched DNA (1mtDNA), and noncomplementary DNA (ncDNA) sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Their base sequences and stock solutions were the same as those in ref 15. Preparation of the Modified Electrodes. The CPE was fabricated using the method reported by Yang et al.16 Graphite oxide (GO) was prepared by the Hummers method.17 SPAN was prepared according to the method reported in ref 18. The preparation of GNO−SPAN composite was carried out by a simple ultrasonic exfoliation and intercalation route based on our previous work.19 In brief, the procedure was as follows: A certain amount of GO was mixed with SPAN at a mass ratio of 1:1. The mixture was diluted to 0.1 g L−1 with ultrapure water and then ultrasonicated for 30 min, giving a suspension of GNO−SPAN composite. Next, 10.0 μL of this suspension was dripped onto a fresh CPE surface and allowed to dry naturally in air. The obtained electrode was denoted as GNO−SPAN/ CPE. The SPAN/CPE was fabricated by a similar procedure, excluding the addition of GO. The preparation routes of the erGNO−SPAN/CPE and trGNO−SPAN/CPE are shown in Scheme 1. The erGNO− SPAN/CPE was fabricated by electrochemical reduction of the GNO−SPAN composite on the CPE, so the GNO−SPAN

electrochemical sensing and biosensing performances of CMGbased composites have rarely been characterized comprehensively. Therefore, further study is needed for both a better understanding of the interaction between CMGs and other components and the future development of high-performance CMG-based composites as nanoscaffolds in electrochemical sensing and biosensing. In this work, three different composites based on CMGs and sulfonic acid-doped polyaniline nanofiber (SPAN, one of the most intensively investigated and widely used conducting polymers), namely, CMG−SPAN composites, were fabricated. The CMGs used here were graphene oxide (GNO), electrochemically reduced graphene oxide (erGNO), and thermally reduced graphene oxide (trGNO). The effects of these CMGs on the morphologies and electrochemical properties of the resulting composites were investigated systematically. Meanwhile, the effects of the three different CMGs on the self-redox reaction activity of SPAN were also studied. To our knowledge, the self-signal change about the redox reaction of SPAN can be quite suitable for the label-free and safe detection of some biomolecules, such as DNA, and improving the self-redox reaction signal of SPAN for highly sensitive DNA detection is a central goal that is worthy of pursuit. Hence, the self-redox reaction signals of SPAN in these CMG-based composites were carefully characterized and used for the rapid, direct, and labelfree detection of DNA. Based on the results, the capabilities of the fabricated CMG−SPAN composite-modified electrodes for DNA analysis were compared. In addition, considering the potential benefits of the trGNO−SPAN composite in supercapacitor area that were surprisingly observed in this research, a preliminary study of the capacitive characteristics of this composite material was conducted. The findings of this work will provide useful guides for future research and applications of CMG−SPAN composites in the fields of electrochemical sensing/biosensing and supercapacitor.

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EXPERIMENTAL SECTION Equipment and Reagents. A CHI 660D electrochemical system (Shanghai CH Instrument Company, Shanghai, China) was employed for the measurement of electrochemical data. The three-electrode system was used throughout, consisting of a carbon paste electrode (CPE), modified CPE, or modified stainless steel sheet as the working electrode; a saturated calomel electrode as the reference electrode; and a platinum wire as the auxiliary electrode. The pH values of all solutions 9077

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Figure 1. SEM images of (A) the SPAN suspension, (B) the SPAN/CPE, and (C−F) CMG−SPAN composites on the CPE surface: (C) GNO− SPAN/CPE, (D) erGNO−SPAN/CPE, and (E,F) trGNO−SPAN/CPE at (E) high and (F) low magnification.

the electrode was thoroughly washed with 0.2% SDS solution before measurements. Electrochemical Measurements. The chronocoulometric measurements were carried out in 2.0 M KCl solution containing 1.0 mM K3[Fe(CN)6]. The parameters were as follows: 0.6 V initial potential, −0.2 V final potential, one single step, and 8-s pulse width. Cyclic voltammograms were recorded in 2.0 M KCl solution containing 1.0 mM K3[Fe(CN)6] and 1.0 mM K4[Fe(CN)6] (denoted as 1.0 mM [Fe(CN)6]3−/4−) and in 0.2 M phosphatebuffered saline (PBS; pH 6.0). In 1.0 mM [Fe(CN)6]3−/4−, the potential scanning range was from 0.6 to −0.2 V at a scan rate of 0.1 V s−1, whereas in 0.2 M PBS, the CV experiment was performed between 0.8 and −0.6 V at a scan rate of 0.05 V s−1. For electrochemical impedance spectroscopy (EIS) measurements, the ac voltage amplitude was +0.005 V, the voltage frequency range was from 105 to 0.1 Hz, and the applied potential was +0.172 V versus saturated calomel electrode (SCE). The supporting electrolyte was 1.0 mM [Fe(CN)6]3−/4−. The calculated results were obtained from the fitting circuit model (Figure 2C inset) with ZSimpWin software. For the capacitive characteristics tests, all electrochemical experiments were conducted in 1.0 M H2SO4 solution. EIS measurements of the trGNO−SPAN supercapacitor were performed from 105 to 0.1 Hz at the open-circuit potential. Charge−discharge measurements were carried out galvanostatically at rates of 0.5−20 A g−1 over a voltage range of 0.8−0 V. The reported results for every electrode in the above assay represent the mean values of three parallel measurements. All experiments were carried out at room temperature.

composite was used as the starting material. Cyclic voltammetry (CV) was used for the electrochemical reduction, and the reduction reaction was performed in the potential range from 0 to −1.7 V at a scan rate of 0.05 V s−1. After a certain number of scan cycles, the erGNO−SPAN/CPE was obtained. For the preparation of the trGNO−SPAN/CPE, an initial thermal reduction step was needed to fabricate the trGNO (as shown in Scheme 1). The obtained trGNO and SPAN were used as the starting materials, and the subsequent procedure was the same as that used for the preparation of the GNO− SPAN/CPE. Preparation of Electrodes for Capacitive Characteristics Tests. A stainless steel sheet was used as the current collector. The electrodes were prepared by the following procedure: Active material, namely, trGNO−SPAN composite obtained from drying the suspension of 0.1 g L−1 trGNO− SPAN composite under a vacuum at 30 °C (75 wt %), was mixed with acetylene black (20 wt %) as a conductive agent, and polytetrafluoroethylene (5 wt %) was dissolved in distilled water as a binder to form a slurry. The paste was then pressed onto the stainless steel sheet at 10 MPa for 1 min to ensure good electrical contact. The modified stainless steel sheet was finally dried under a vacuum at 70 °C for 24 h. Immobilization and Hybridization. Based on the interaction between the amine groups of pDNA and the sulfonic acid groups of SPAN,20 pDNA could be covalently immobilized on the CMG−SPAN/CPE surfaces. Before the immobilization, the modified electrode was immersed in an acetone solution containing 40.0 mM PCl5 for 30 min to activate the sulfonic acid groups. After activation, the modified electrode was rinsed with Tris-NaCl buffer solution (50.0 mM Tris-HCl, 50.0 mM NaCl, pH 7.0) to wash off the excess PCl5. Then, 20.0 μL of Tris-NaCl buffer solution containing 1.0 × 10−6 M pDNA was dropped onto the CMG−SPAN/CPEs and allowed to dry naturally in air. Before electrochemical analysis, the pDNA-modified electrodes were washed extensively with ultrapure water to remove the unimmobilized oligonucleotides. To achieve hybridization of the elctrodes with cDNA, 1mtDNA, or ncDNA, 20.0 μL of hybridization solution [2 × saline−sodium citrate (SSC) buffer, pH 7.0] containing a certain amount of cDNA, 1mtDNA, or ncDNA was dropped onto a probe-modified electrode, and the hybridization reaction was allowed to proceed for a hybridization time of 1 h. Then,

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RESULTS AND DISCUSSION Morphology Characterization of the SPAN and CMG− SPAN Composites. Figure 1 shows SEM images of the SPAN and CMG−SPAN composites. It can be seen that the SPAN suspension was well-dispersed and presented a network structure (Figure 1A). Once modified on the CPE, the SPANs agglomerated together and formed an undesirable aggregated structure on the electrode surface (Figure 1B).21 In contrast, as a result of the modification of the ultrasonically treated GNO−SPAN composite, one can clearly observe the structure of wrinkled GNO intercalated with well-dispersed 9078

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The Journal of Physical Chemistry C SPANs on the electrode surface (Figure 1C), suggesting that GO was well-exfoliated22,23 and that full intercalation between the GNO and SPANs occurred during the process of ultrasonic treatment. These results are consistent with those of our previous experiments19 and illustrate that the formation of GNO−SPAN composites is an effective way to prevent the agglomeration of SPANs and the restacking of GNO. As shown in Figure 1D, the morphology of the erGNO−SPAN composite was quite similar to that of the GNO−SPAN composite on the electrode surface. This might be because the erGNO−SPAN composite was fabricated using the GNO−SPAN composite as the starting material and the subsequent reduction did not induce changes in the morphology.10 However, for the trGNO−SPAN composite film (Figure 1E,F), the morphology was found to be dramatically different. Compared with the GNO−SPAN and erGNO−SPAN composites, the trGNO− SPAN composite had fewer wrinkles and a relatively smooth and compact surface. This might be due to the restacking of the trGNO and poor exfoliation of the stacked trGNO under the ultrasonic conditions that we used. In addition, it is notable that the SPANs, which were intercalated between the trGNO layers, had a lower density than in the other two composites. Instead, some SPANs were adsorbed on the outer surface of the trGNO, providing further evidence that the prepared trGNO was difficult to fully exfoliate under the conditions employed. The low-level exfoliation of trGNO and the relatively poor intercalation of SPANs are not conducive to increasing the electrode surface area. However, the low-magnification SEM image in Figure 1F shows that the trGNO−SPAN composite film presented a unique interconnected macroporous network. This porous structure can reduce the impact of the above factors and help to enlarge the electrode surface area.24 The above results indicate that GO is more easily exfoliated than trGNO under the same ultrasonic conditions, probably because of the higher coverage of oxygen-containing functional groups on the basal plane of the former, which results in electrostatic repulsion between the GO layers and increases the interlayer distance.25 Therefore, the morphology of the CMG−SPAN composites is mainly dependent on the preparation procedures (intercalation first or reduction first). When the GNO−SPAN composite was formed first, the morphology of the product was not significantly affected by the subsequent reduction. Electrochemical Characterization of the Modified Electrodes. Figure 2A presents chronocoulometric curves at different modified electrodes for the reduction of 1.0 mM [Fe(CN)6]3−. After subtraction of the background charge, linear relationships between the charge Q (from reduction of [Fe(CN)6]3− diffusing from the solution) and the square root of time (t1/2) were obtained at all four of the modified electrodes, namely, SPAN/CPE, GNO−SPAN/CPE, erGNO− SPAN/CPE, and trGNO−SPAN/CPE, in accordance with the equation Q = (2nFAD01/2π −1/2C0)t 1/2

Figure 2. (A) Chronocoulometric curves for the reduction of 1.0 mM [Fe(CN)6]3−, (B) CV curves in 1.0 mM [Fe(CN)6]3−/4−, and (C) Nyquist plots in 1.0 mM [Fe(CN)6]3−/4− for the following electrodes: (a) SPAN/CPE, (b) GNO−SPAN/CPE, (c) erGNO−SPAN/CPE, and (d) trGNO−SPAN/CPE. In panel C, the experimental data are shown as symbols, and the fits are shown as solid lines. Inset: Equivalent circuit model.

one can see that the highest value of A was observed at the GNO−SPAN/CPE, but the difference between the areas of the GNO−SPAN/CPE and erGNO−SPAN/CPE was not significant. This can be attributed to their similar morphologies.10 The tiny difference, however, might be due to the small change in the interlayer force between the functional graphene nanosheets caused by the removal of the oxygen-containing groups after reduction. Compared with the GNO−SPAN/CPE and erGNO−SPAN/CPE, the trGNO−SPAN/CPE exhibited a significantly smaller apparent electrode area, because of the low-level exfoliation of trGNO and the relatively poor intercalation of SPANs. The SPAN-modified electrode had the smallest apparent electrode area, owing to the agglomer-

(1)

where n is the number of electrons in the reaction, F is the Faraday constant, A is the apparent electrode area, D0 is the diffusion coefficient of [Fe(CN)6]3−, and C0 is the bulk concentration of [Fe(CN)6]3−. The apparent electrode area A can be calculated from the slope of the Q−t1/2 linear curve. The order of the A values for the different electrodes was found to be GNO−SPAN/CPE (0.526 ± 0.015 cm2) > erGNO−SPAN/ CPE (0.488 ± 0.013 cm2) > trGNO−SPAN/CPE (0.383 ± 0.020 cm2) > SPAN/CPE (0.233 ± 0.001 cm2). From the data, 9079

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further confirmation that the trGNO−SPAN compositemodified electrode exhibits a capacitive property. The capacitive characteristics of the trGNO−SPAN composite were investigated toward the end of this work to explore its potential applications in supercapacitors. To gain quantitative EIS information about the studied electrode materials, the Randles equivalent circuit (inset of Figure 2C) was chosen to fit the obtained impedance spectra. The equivalent circuit includes the ohmic resistance, Rs, of the electrolyte solution and the electronic charge-transfer resistance, Rct, in series with the Warburg impedance, W, and in parallel with the constant phase element. The use of a constant phase element instead of a capacitor was required to achieve a better fitting of the experimental data, because a constant phase element can reflect the properties of these nonideal electrode surfaces that exhibit depressed semicircles and non-90° capacitive lines.29 Based on the equivalent circuit, the value of Rct was calculated to be 3364, 2166, 56.23, and 13.55 Ω·cm2, respectively, for the SPAN/CPE, GNO−SPAN/CPE, erGNO− SPAN/CPE and trGNO−SPAN/CPE. The calculated result is in good agreement with the experimental results shown in Figure 2B,C and provides direct confirmation that (1) the integration of CMG with SPAN, instead of SPAN alone, can improve the charge-transfer performances of the modified electrodes, especially for the rGNO−SPAN compositemodified electrodes, because of the significantly improved electrical conductivity of the rGNO component, and (2) the trGNO−SPAN composite presents higher electrical conductivity than the erGNO−SPAN composite. To our knowledge, the electrical conductivity is a good indicator of the extent to which GNO has been reduced.30 Therefore, the trGNO employed here could be reduced to a greater degree than the erGNO. Moreover, the n value of the constant phase element for the trGNO−SPAN/CPE is 0.81, indicating the capacitative character of the interface trGNO−SPAN composite film/ solution,31 which is also consistent with the obtained experimental results. Redox Behaviors of SPAN. Because the self-redox signal of SPAN has a potential application in serving as the measurement signal for developing the rapid, direct, and label-free DNA detection assay,32 the redox behaviors of SPAN in the above composites were studied. Figure 3 shows the redox behaviors of SPAN during CV in 0.2 M PBS (pH 6.0) between 0.8 and −0.6 V. Because of the presence of electroactive SPAN,

ation of the nanofibers. These results indicate that the addition of CMG greatly improved the apparent electrode areas of the modified electrodes, because of the better dispersion of the SPANs in these composites and the large surface areas of the functional graphene nanosheets. Meanwhile, the results further confirm that the morphology of the GNO−SPAN composite was not significantly affected by the subsequent electroreduction reaction. To further characterize the electrochemical properties of these composite-modified electrodes, their performances toward the [Fe(CN)6]3−/4− redox couple were investigated. From the CV curves shown in Figure 2B, marked differences in the electrochemical activities among SPAN/CPE, GNO− SPAN/CPE, erGNO−SPAN/CPE, and trGNO−SPAN/CPE can be recognized. In detail, a pair of weak redox peaks of [Fe(CN)6]3−/4− was observed for the SPAN/CPE (curve a). By contrast, the redox peak currents of [Fe(CN)6]3−/4− increased dramatically for the GNO−SPAN/CPE (curve b). The enhanced electrochemical activity of the GNO−SPAN/CPE was probably caused by the synergistic effect between GNO and SPAN, which can be ascribed to the larger electrode surface area caused by the better dispersion of SPAN and GNO and the higher charge-transfer rate resulting from the tight intercalation between GNO and SPAN. Compared with those of the SPAN/CPE and GNO−SPAN/CPE, the electrochemical responses of the redox couple at the erGNO−SPAN/CPE and trGNO−SPAN/CPE further increased, owing to the reduction of the surface negative charges and the improved electrical conductivity of the rGNO.23 In addition, it is notable that the CV curve of the trGNO−SPAN/CPE had a large rectangular area, indicating a large electric double-layer capacitance on this composite-modified electrode that can be ascribed to its unique porous structure,26 as confirmed by the SEM images (Figure 1F) and high electrical conductivity. The EIS measurements were analyzed using Nyquist plots to obtain more detailed information about the interface properties of the SPAN- and composite-modified electrodes. The typical Nyquist plots are presented in Figure 2C. The impedance spectra at both the SPAN/CPE (curve a) and GNO−SPAN/ CPE (curve b) are composed of depressed semicircular arcs in the high-frequency region and straight lines in the lowfrequency region. The large semicircular arcs in curves a and b demonstrate the high interfacial charge-transfer resistances of the two modified electrodes,27 which can be attributed to the aggregation of the SPANs and poor conductivity of the GNO. In contrast to the results for the SPAN/CPE and GNO− SPAN/CPE, semicircular arcs in the high-frequency region are unnoticeable for the erGNO−SPAN/CPE (curve c) and trGNO−SPAN/CPE (curve d), suggesting that the interfacial charge-transfer resistances of the erGNO−SPAN/CPE and trGNO−SPAN/CPE are very low, because of the improved electrical conductivity of the rGNO. In addition to the differences in the high-frequency region, the difference in the low-frequency region between the first two and last two electrodes is also significant. As shown, the Nyquist plots of the erGNO−SPAN/CPE and trGNO−SPAN/CPE show higher slopes than those of the SPAN/CPE and GNO−SPAN/CPE in the low-frequency linear region. Especially for the trGNO− SPAN/CPE, the line obviously deviates from the ideal 45° slant of the classical Warburg diffusion line. It is well-known that a phase shift angle of impedance to 90° represents ideal capacitor charging. The more vertical the line, the higher capacitance performance of the electrode.28 Hence, our results provide

Figure 3. CV curves of (a) SPAN-, (b) GNO−SPAN-, (c) erGNO− SPAN-, and (d) trGNO−SPAN-modified electrodes in 0.2 M PBS (pH 6.0). 9080

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The Journal of Physical Chemistry C the CV curves of the four electrodes all display two pairs of redox peaks (C1/A1, C2/A2), which can be ascribed to the well-known leucoemeraldine (LE)/emeraldine (EB) and emeraldine (EB)/pernigraniline (PNB) redox transitions.33,34 Compared with those of the SPAN-only-modified electrode, the current response and CV loop area of the CMG−SPANmodified electrodes were all increased. These results indicate that the presence of GNO or rGNO can enhance the redox activity of the SPAN. This might be because the SPAN can achieve better dispersion in these composites and the rGNO can form highly conductive networks that, individually or collectively, can improve the redox activity of SPAN. Taking the peak current of C1 as the measurement signal, the sequence of the values for different composite-modified electrodes was found to be trGNO−SPAN/CPE > erGNO−SPAN/CPE > GNO−SPAN/CPE, which indicates that different CMGs have different effects on the redox activity of SPAN. This sequence has the same tendency as the charge-transfer rate of these composite-modified electrodes, shown in Figure 2C. These results demonstrate that the more conductive the network formed by CMG, the greater the enhancement of the redox activity of SPAN. The composite with trGNO had the highest electrical conductivity; thus, the redox activity of SPAN in this composite was improved by the greatest degree. Label-Free Electrochemical Detection of DNA Immobilization and Hybridization. After the electrochemical characterization and optimization of the preparation conditions (see the Supporting Information, Figure S1), the GNO−SPAN, erGNO−SPAN, and trGNO−SPAN composites were used as electrode materials for constructing electroactive platforms for DNA sensing. The CV curves were recorded (Figure 4), and the self-redox signal changes of the SPAN in the composites after DNA immobilization and hybridization were used to detect the PML/RARA fusion gene segment. The difference in the reduction peak current of peak C1 (namely, ΔIpc) between the probe-captured electrode and its cDNA hybridizationinduced electrode was used as the measurement signal. As shown in Figure 4A, after covalent immobilization of pDNA, the peak current of SPAN on the GNO−SPAN/CPE obviously decreased. This phenomenon also occurred on the erGNO− SPAN/CPE and trGNO−SPAN/CPE (Figure 4B,C) and can be attributed to the impaired counterion exchange caused by the introduction of the DNA bulky chains and their negative charge.35,36 After hybridization with cDNA, further decreases of the signal were observed on the three electrodes. The decrease of the current values might be due to the addition of negative charges to the electrode surface after cDNA hybridization, further suppressing the redox reaction of the SPAN37,38 and suggesting that the resulted dsDNA remains on the sensing platform after hybridization.36,39 The peak current decreased gradually with increasing target sequence concentration. The ΔIpc value and the logarithm of the PML/RARA gene sequence concentration all showed linear relationships in a certain range of gene sequence concentrations (Figure 5A) at the three modified electrodes. The slopes of the lines were used to evaluate the sensitivities of the biosensors.40 The specificity of the CMG−SPAN/CPEs for label-free detection of DNA hybridization was also investigated by measuring changes in the electrochemical current that occurred upon hybridization with different DNA sequences (including 1mtDNA and ncDNA). As illustrated in Figure 5B, for all the investigated electrodes, the largest signal decreases were observed for the cDNA sequences, whereas lower signal

Figure 4. CV curves of the (A) GNO−SPAN/CPE, (B) erGNO− SPAN/CPE, and (C) trGNO−SPAN/CPE in 0.2 M PBS (pH 6.0): before the immobilization of pDNA, after the immobilization of pDNA, and after the hybridization reaction with different concentrations of PML/RARA fusion gene sequence [for A, (a) 10−15, (b) 10−14, (c) 10−12, (d) 10−10, (e) 10−8, and (f) 10−7 M; for B,C, (a) 10−16, (b) 10−15, (c) 10−14, (d) 10−12, (e) 10−10, (f) 10−8, and (g) 10−7 M].

changes were seen for the 1mtDNA sequences, and the signal changes observed for the ncDNA sequences were the smallest and negligible. This demonstrates that all the three label-free electrochemical biosensors based on CMG−SPAN composite sensing platforms exhibit good selectivity. The sensitivity, log−linear range, detection limit, and square of the correlation coefficient (R2) were calculated and are presented in Table 1. It can be seen that the erGNO−SPAN and trGNO−SPAN composite-modified electrodes showed higher detection sensitivities, wider linear ranges, and lower detection limits than the GNO−SPAN/CPE. This can be 9081

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SPAN composite was studied for its application in the supercapacitor field because of its potential benefits in supercapacitors that were revealed in this research. A preliminary study to investigate its capacitive characteristics was conducted. For details, see the Supporting Information (Figure S2). The results show that the trGNO−SPAN composite material exhibits excellent capacitive performances [a high specific capacitance of 603 F g−1 at a current density of 0.5 A g−1, as well as a capacitive retention of 62% when the current density was increased in a wide range (from 0.5 to 20 A g−1)], which can be attributed to the contributions of both electric double-layer capacitance and pseudocapacitance, together with the high electrical conductivity of the composite. These performances make the trGNO−SPAN composite a promising electrode material for supercapacitors, which have wide applications in electric vehicles and other fields.

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CONCLUSIONS In this work, the effects of various CMGs on the morphologies, electrochemical properties, and DNA sensing performances of CMG−SPAN composites were comprehensively studied. We observed that GO is easier to exfoliate than trGNO under the same ultrasonic conditions, leading to the conclusion that the morphology of the resulting composite is greatly affected by the preparation procedure. The electrochemical performances of these CMG−SPAN composite-modified electrodes all improved in comparison with that of the SPAN-only-modified electrode, and both the open structure of the CMG−SPAN composites and the highly conductive networks formed by rGNO obviously enhanced the redox activity of SPAN. Based on the significantly enhanced self-redox signals of SPAN in these composites, the rapid, direct, and label-free DNA detection was successfully realized. In comparison with the GNO−SPAN composite-modified electrode, the rGNO− SPAN composite-modified electrodes exhibited a higher sensitivity, a wider linear range, and a lower detection limit. These results indicate that differences in the surface functional groups, structures, and conductivities among the various CMGs have a great influence on the morphologies, electrochemical properties, and DNA analysis capacities of the CMG−SPAN composites. In addition, the capacitive characteristics of the trGNO−SPAN composite were investigated further, and excellent capacitive performances of the composite material were found. We believe that the findings of this work will provide useful guides for future research and applications of CMG−SPAN composites in the areas of electrochemical sensing/biosensing and even supercapacitors.

Figure 5. (A) Plot of ΔIpc vs the logarithm of the PML/RARA fusion gene segment concentration for the GNO−SPAN/CPE, erGNO− SPAN/CPE, and trGNO−SPAN/CPE. (B) Comparison of hybridization signal changes for pDNA modification of the same electrodes after hybridization with cDNA, 1mtDNA, and ncDNA. The concentrations of these sequences were all selected as 1.0 × 10−7 M.

attributed to the higher electrical conductivity of these two rGNO−SPAN composite-modified electrodes, which greatly promotes the self-redox signal of SPAN, and the fact that the excellent self-redox response can sensitively monitor the changes induced by hybridization with the target sequences. The two rGNO−SPAN/CPEs had the same log−linear range, but the erGNO−SPAN/CPE exhibited a higher detection sensitivity and a lower detection limit than the trGNO−SPAN/ CPE. This is inconsistent with our expectations; it is likely that the morphology and structure of the erGNO−SPAN composite on the electrode are conducive to hybridization by facilitating the display of probe sequences on the nanostructure that are in favor of capturing target molecules.41 The reproducibility of the CV measurements on the composite-modified electrodes was also studied. The relative standard deviation (RSD) from the slopes of three calibration plots was calculated. For the GNO−SPAN/CPE, erGNO− SPAN/CPE, and trGNO−SPAN/CPE, the RSDs were 15.06%, 8.65%, and 13.09%, respectively. From these data one can see that DNA biosensors based on these three composites all showed good reproducibility and that the CV measurements on the erGNO−SPAN/CPE were the most reproducible among the three electrodes. Extended Application of the trGNO−SPAN Composite. In addition to the above investigations, the trGNO−

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ASSOCIATED CONTENT

S Supporting Information *

Optimization of the reduction conditions (Figure S1) and capacitive characteristics of the trGNO−SPAN composite (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

Table 1. Sensitivity, Log−Linear Range, Detection Limit, and Square of the Correlation Coefficient (R2) for the Determination of the PML/RARA Gene Sequence on Different CMG−SPAN Composite-Modified Electrodes electrode

sensitivity [μA (log M)−1]

log−linear range (M)

detection limit (M)

R2

GNO−SPAN/CPE erGNO−SPAN/CPE trGNO−SPAN/CPE

1.165 4.762 4.150

1.0 × 10−15−1.0 × 10−7 1.0 × 10−16−1.0 × 10−7 1.0 × 10−16−1.0 × 10−7

2.75 × 10−16 3.22 × 10−17 6.56 × 10−17

0.9880 0.9924 0.9941

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are most grateful to the National Natural Science Foundation of China (Nos. 21275084, 41476083), Doctoral Foundation of the Ministry of Education of China (No. 20113719130001), Scientific and Technical Development Project of Qingdao [No. 12-1-4-3-(23)-jch], and Outstanding Adult-Young Scientific Research Encouraging Foundation of Shandong Province (No. BS2012CL013).

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