Graphene-palladium nanowires based electrochemical sensor using ZnFe2O4-graphene quantum dots as an effective peroxidase mimic.

G Model ACA 233454 No. of Pages 8

Analytica Chimica Acta xxx (2014) exxx–exxx

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Graphene–palladium nanowires based electrochemical sensor using ZnFe2O4–graphene quantum dots as an effective peroxidase mimic Weiyan Liu a , Hongmei Yang a , Chao Ma a , Ya-nan Ding a , Shenguang Ge b , Jinghua Yu a , Mei Yan a, * a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, PR China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

The nanohybrid ZnFe2O4/GQDs was developed by assembling the GQDs on the ZnFe2O4 through a photoFenton reaction. The ZnFe2O4/GQDs exhibited higher peroxidase-like activity and better stability than each individual and HRP. An electrochemical sensor was fabusing ZnFe2O4/GQDs ricated nanohybrid as a mimic enzymatic to detect DNA. Graphene and Pd nanowires were modified on the glassy carbon electrode, which improved the electronic transfer rate.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 June 2014 Received in revised form 22 August 2014 Accepted 27 August 2014 Available online xxx

We proposed an electrochemical DNA sensor by using peroxidase-like magnetic ZnFe2O4–graphene quantum dots (ZnFe2O4/GQDs) nanohybrid as a mimic enzymatic label. Aminated graphene and Pd nanowires were successively modified on glassy carbon electrode, which improved the electronic transfer rate as well as increased the amount of immobilized capture ssDNA (S1). The nanohybrid ZnFe2O4/GQDs was prepared by assembling the GQDs on the surface of ZnFe2O4 through a photo-Fenton reaction, which was not only used as a mimic enzyme but also as a carrier to label complementary ssDNA (S3). By synergistically integrating highly catalytically activity of nano-sized GQDs and ZnFe2O4, the nanohybrid possessed highly-efficient peroxidase-like catalytic activity which could produce a large current toward the reduction of H2O2 for signal amplification. Thionine was used as an excellent electron mediator. Compared with traditional enzyme labels, the mimic enzyme ZnFe2O4/GQDs exhibited many advantages such as environment friendly and better stability. Under the optimal conditions, the approach provided a wide linear range from 1016 to 5 109 M and low detection limit of 6.2 1017 M. The remarkable high catalytic capability could allow the nanohybrid to replace conventional peroxidase-based assay systems. The new, robust and convenient assay systems can be widely utilized for the identification of other target molecules. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Mimic enzymes ZnFe2O4–graphene quantum dots composites Electrochemical sensor Palladium nanowires

* Corresponding author. Tel.: +86 531 82767161. E-mail address: [email protected] (M. Yan). http://dx.doi.org/10.1016/j.aca.2014.08.054 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: W. Liu, et al., Graphene–palladium nanowires based electrochemical sensor using ZnFe2O4–graphene quantum dots as an effective peroxidase mimic, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.08.054


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W. Liu et al. / Analytica Chimica Acta xxx (2014) xxx–xxx

1. Introduction The specific sequence detection of DNA has attracted considerable interest due to its broad applications in molecular diagnostics, genetic therapy and early screening of diseases [1]. Conventional detection methods for DNA including optical method [2], quartz–crystal microbalance [3] and surface plasmon resonance techniques [4] not only need sophisticated instrumentations but suffer from radiation hazards and long analysis times [5]. Electrochemical assays are the important analytical tools in clinical analyses, because they are simple instrumentation and operation, and provides fast analysis [6]. Enzyme based electrochemical assay as one of the most common used methods has the advantages of high sensitivity and good selectivity. However, natural enzymes are easily affected by environmental changes such as pH and temperature, furthermore, they suffer from difficulty in preparation and purification. To resolve this issue, much attention has been focused on developing enzyme mimetics. Various materials were found to possess intrinsic peroxidase-like activity and have been used in various fields such as H2O2 detection [7], cytotoxicity assay [8] and glucose detection [9]. These enzyme-like nanomaterials were of low cost, easy preparation, high stability in comparison with HRP. Thus, NPs enzyme mimetics have great potential applications in medical diagnosis and biosensing [10]. However, the application of some nanomaterials was restricted due to difficulty in separation and instability [11]. ZnFe2O4 MNP, one of the spinel ferrite compound, were more stable with exposure to air and could be separated by a magnet [12]. It has been attracted considerable attention in the conversion of solar energy, and photochemical hydrogen production from water due to its visible-light response, good stability, and low cost [13,14]. It also possessed intrinsic peroxidase-like catalytic activity and has been used in colorimetric biosensor for detection of urine glucose [12]. However, few papers reported their application in electrochemical sensors as a mimic enzyme. In addition, the peroxidase-like activities of bare ZnFe2O4 MNPs are relatively low [15]. Therefore, further investigation to design and fabricate ZnFe2O4 based hybrid materials which could provide enhanced catalytic performance and stability are required for its wide-ranging applications. Graphene quantum dots (GQDs), the graphene sheets with lateral size less than 100 nm, have several unique properties over micrometer-sized graphene and graphene oxide (GO) [16]. Due to quantum confinement and edge effects, GQDs presented fascinating properties, such as strongly luminescent, eco-friendly and excellent biocompatibility, and that make it applied in bioimaging and photovoltaic devices [17]. However, the study of the catalytic activity of GQDs is rare. The GQDs, prepared through the photo-Fenton reaction of GO have intrinsic peroxidase-like activities. The catalytic property is much higher than the micrometer sized GO, as they have large specific surface area, defect-free aromatic structure and rich periphery carboxylic groups [18]. Another reason is that the size change can efficiently improve the specific surface area and distinct crystal sizes contain different atomic arrangements [19]. However, so far relatively little attention has been paid to the catalysis of composite catalyst. It is of great interest to study. In this paper, ZnFe2O4/GQDs composites were successfully prepared by in situ controlled nucleation of GQDs on ZnFe2O4. The resultant nanocomposites showed both enhanced peroxidase-like activity than each of component and magnetism. The complex artificial enzymes have several advantages such as easy preparation, low-cost, and high stability and high catalytic property. In recent years, several nanomaterials [20,21] were used to modify electrochemical interface to improve the sensitivity because they could act as enhanced elements for effectively

accelerating the electron transfer between electrode and probe molecules and provide a very high electrochemically active area [22]. Graphene sheet (GS) as a 2D single layer of sp2 hybridized carbon atoms with the hexagonal honeycomb lattice has been widely used [23]. However, GS is insoluble and hard to disperse in all solvents [24]. Here, we introduced a primary amine into GS to improve the solubility (the aminated graphene was also named GS below). In addition, Pd nanowires (NWs) are less vulnerable and Ostwald ripening and can provide higher electrochemically active area for target molecules than Pd NPs due to their micrometer-sized length. [25]. Here, we construct a Pd NWs/GS platform via self assemble relying on the electrostatic interaction or interaction between NH2 group and Pd NWs. This Pd NWs/GS possessed higher electron transfer rate as well as higher surface area than each of component due to the synergic effect. We used it to modify glassy carbon electrode (GCE) for immobilization capture of ssDNA (S1). The ZnFe2O4/GQDs mimic was used as a trace label and as a carrier for complementary ssDNA (S3). Based on this, a sensitive and accurate electrochemical DNA sensor was developed. Compared with traditional enzyme labels, the enzyme-like ZnFe2O4/GQDs had unique advantages of being environment friendly and had better stability. 2. Reagents and instruments 2.1. Reagents All reagents were of analytical-reagent grade or the highest purity available and directly used for the following experiments without further purification. The aqueous solutions unless indicated were prepared with ultrapure water and 6-mercapto-1-hexanol (MCH) were purchased from Nanoport. Co., Ltd. (Shenzhen, China). N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar China Ltd. 3,30 ,5,50 -Tetramethylbenzidine (TMB), thionine (TH) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Palladium(II) chloride (PdCl2), poly(vinylpyrrolidone) (PVP), sodium iodide (NaI), hydrazine solution (80 wt%) and ammonia solution (25 wt%) were purchased from Sinopharm Chem. Re. Co., Ltd. (Shanghai, China). The buffers involved in this work are as follows: DNA immobilization buffer, 10 mM Tris–HCl, 1.0 mM EDTA, 1.0 M NaCl, and 1.0 mM TCEP (pH 7.4); DNA hybridization buffer, 10 mM Tris–HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.4). Washing buffer, 10 mM phosphate buffer (PBS), and 0.1 mM NaCl (pH 7.4). All of the synthetic oligonucleotides were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Their base sequences are as follows: Capture probe ssDNA sequence (S1): 50 -TGG AAA ATC TCT AGC AGT CGT-(CH2)6-SH-30 . Target ssDNA sequence (S2): 50 -ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA-30 . Complementary ssDNA sequence (S3): 50 -NH2-(CH2)6-ATG TCC CTC AGA CCC TTT-30 . Two-base mismatched ssDNA sequences (S4): 50 -ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GCG TCT GTG GGA-30 . Non-complementary ssDNA sequences (S5): 50 -ACT GCT AGA GAT TTT CCA CAC TGA CTA CTT CAA CAG TGC CCC-30 . 2.2. Fabrication of ZnFe2O4/GQDs The ZnFe2O4 was synthesized according to the previous literature [12]. A mixture containing 0.34 g of ZnCl2 and 1.35 g of FeCl36H2O was added to 40 mL ethylene glycol and sonicated for 30 min at room temperature to form a clear solution. Then 3.6 g of NaAc and 1.0 g of polyethylene glycol-4000 were added and stirred vigorously for 30 min, yielding a stable bottle-yellow




homogeneous emulsion then sealed in a Teflon-lined stainless-steel autoclave and heated to 200 C for 8 h, followed by cool to room temperature. The black products were washed several times with ethanol and dried at 60 C. The ZnFe2O4/GQDs were prepared as follows: 0.005 g of ZnFe2O4, 5.0 mL of 0.5 mg mL1 GO aqueous suspension, 0.5 mL of 30% H2O2 and 80 mL of 3.0 mM of FeCl3 were mixed in a beaker under vigorous stirring and the pH of the mixture was adjusted to 4.0. The reaction was carried out under vigorous stirring by exposing the beaker to a mercury lamp (365 nm, 1000 W). After reaction, the precipitates were collected by centrifugation and further washed with water to remove the iron ions, trace H2O2, and other small molecular reaction products. Finally, the obtained ZnFe2O4/GQDs were dispersed in water and stored in 4 C for further use.

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The modified working electrode was immediately immersed into immobilization buffer containing 108 M S1 for 80 min then washed to remove physical adsorbed S1. The capture probe modified electrode was further treated with 1.0 mM MCH for 30 min to obtain a well-aligned DNA monolayer and block the uncovered electrode surface, followed by washing with the washing buffer. Subsequently, for the hybridization reaction, the electrode was coated with different concentrations of target DNA (S2) for 80 min at 42 C. Finally, the electrode was secondly hybridized with ZnFe2O4/GQDs-S3 composites for 80 min at 42 C. After hybridization, the DNA sensor was extensively rinsed with washing buffer and dried. The differential pulse voltammetry (DPV) behavior of the DNA sensors was recorded in pH 7.4 Tris–HCl solution containing 0.2 mM H2O2 and 0.1 mM thionine from 0.2 to 0.7 V and the signals related to the S2 concentrations were measured.

2.3. Preparation of ZnFe2O4/GQDs conjugated DNA (S3) 3. Results and discussion ZnFe2O4/GQDs-S3 composite solution was obtained by mixing ZnFe2O4/GQDs, EDC and S3 (1:1:3 volume ratio). The mixture was briefly stirred for 2 h, followed by repeatedly washing three times to remove unspecific physical adsorption. The reaction was based on the interaction between NH2 or COOH groups on the S3 and ZnFe2O4/GQDs. 2.4. Fabrication of the DNA sensor and measurement procedure The fabrication process of the proposed DNA sensors is shown in Scheme 1. The GCE (3 mm diameter) was firstly polished with 0.3 and 0.05 mm alumina slurry, respectively, followed by sonication in ethanol and drying in air. After that, 5 mL GS (the synthetic steps are shown in Supporting information) (1.0 mg mL1) was initially deposited on the electrode surface, and then dried at room temperature. 5 mL of Pd NWs (the synthetic steps are shown in Supporting information) (1.0 mg mL1) were casted on an electrode and dried at room temperature.

3.1. Characterization of ZnFe2O4/GQDs The morphologies of the as-prepared GQDs, ZnFe2O4 and ZnFe2O4/GQDs composite were characterized with transmission electron microscope (TEM) as shown in Fig. 1. We could clearly see that the diameters of GQDs were 5–7 nm from the TEM image (Fig. 1A). The obtained ZnFe2O4 was spherical and dispersive with an average size of about 80–100 nm (Fig. 1B). After assemble of GQDs, the resulting composites were spherical particles with diameter of about 100–120 nm and a slight thin layer on the particles surface (20 nm) were found. This diameter increment and the morphology changes indicated that the GQDs were successfully coated on ZnFe2O4 as shown in Fig. 1C. To get insight into the hyperfine chemical structure, the FTIR spectra of GQDs were acquired and shown in Fig. 1D. For ZnFe2O4, the two strong absorption peaks at lower frequency (around 582 and 453 cm1) could be assigned to the stretching vibrations of

Scheme 1. Schematic illustration of ZnFe2O4/GQDs as a mimicking trace label for electrochemical detection of DNA.



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Fig. 1. TEM images of GQDs (A), ZnFe2O4 (B), ZnFe2O4/GQDs (C). FTIR spectrum of ZnFe2O4 (a) GQDs (b) ZnFe2O4/GQDs (c) (D).

the Zn O bonds in tetrahedral positions and the Fe O bonds in octahedral positions. For GQDs in curve b, the peak located at 1721 cm1 could be attributed to characteristic bands of the CQO stretching vibrations of COOH groups, the band at 1227 cm1 was attributed to C O (epoxy) stretching vibration, the deformation peaks at around 1389 cm1 was attributed to OH groups and the bonds at 1564 cm1 was corresponded to CQC. The results indicated that the surfaces of the GQDs were full of carboxy groups. After the formation of ZnFe2O4/GQDs, a new peak was observed at 1628 cm1, but the characteristic bands of the CQO stretching vibrations of COOH groups at 1721 cm1 also existed, specifying that a part of the periphery carboxy groups of GQDs were converted into carboxylate. This result indicated that the GQDs bind to the ZnFe2O4 particles through the Fe O chemical bonds most probably. Moreover, the characteristic bands of Zn O and FeO were blue-shifted to 536 and 425 cm1 which further demonstrated the formation of the nanohybrid. Fig. S1 shows the typical X-ray diffraction profiles of GQDs, ZnFe2O4 and ZnFe2O4/GQDs. The GQDs have a broader (0 0 2) peak centered at around 21.5 in curve a. In curve b, the peaks at 2u values of 30.1, 35.2, 42.8, 53.2, 56.8, and 62.4 could be well-assigned to the (2 2 0), (3 11), (4 0 0), (4 2 2), (5 11), and (4 4 0) crystal planes of spinel ZnFe2O4, respectively. All the diffraction peaks can be directly indexed to the spinel-type ZnFe2O4 (JCPDS 22-1012). But in curve c, it can be seen that the typical diffraction peaks of ZnFe2O4 were weak or disappeared and the diffraction peaks of GQDs were observable. This may be ascribed to the fact that the ZnFe2O4 were exfoliated by decorating GQDs.

3.2. The peroxidase-like catalytic activity of ZnFe2O4/GQDs To investigate the peroxidase-like catalytic activity of the ZnFe2O4/GQDs, the ZnFe2O4/GQDs-catalyzed reaction of peroxidase substrate TMB with H2O2 was tested. The activity was also compared with those of bare GQDs and ZnFe2O4. As shown in the inset of Fig. 2A, the ZnFe2O4/GQDs can catalyze the oxidation of TMB by H2O2 to produce the typical blue color and the color was much deeper than each of component and could observed just by the naked-eye. The results were further confirmed with the UV–vis absorption (Fig. 2A). We can see that the as-obtained ZnFe2O4/GQDs composite (curve c) had much higher absorption than others (curve a for ZnFe2O4, curve b for GQDs) under the same reaction conditions. In addition, the catalytic performance of the proposed catalyst was compared with commercial HRP by comparing the formation of reaction products against time after the addition of the catalysts (the same amount of HRP and ZnFe2O4/GQDs was used) as shown in Fig. 2B. The color change was very fast in the presence of ZnFe2O4/GQDs, which was completely achieved within only 6 min (curve a). However, the color changed slightly during the 6 min for HRP catalysts (curve b). The result showed that ZnFe2O4/GQDs possess superior peroxidase-like catalytic performance than HRP. The reason for such high catalytic activity may be due to the synergistic effect of individual, not a simple addition of the activities of ZnFe2O4 and GQDs. The formed ZnFe2O4/GQDs conjugates make the electron-transfer from the electron rich GQDs to ZnFe2O4 more efficient. Moreover, the other reason was attributed to the small




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Fig. 2. (A) UV–vis absorption spectra of TMB–H2O2 mixed solution in ZnFe2O4 (a) GQDs (b) and ZnFe2O4/GQDs (c). Experimental conditions: 20 mM TMB, 0.2 mM H2O2, 0.2 mM acetate buffer (pH 4.0). The inset of (A) is the images of (a–c). (B) Time-dependent absorbance changes at 652 nm of TMB in ZnFe2O4/GQDs (a) and HRP (b) catalysis. Catalytic stabilities of ZnFe2O4/GQDs (a) and HRP (b) against (C) pH and (D) temperature. In Fig. 3B–D, the error bars represent the standard deviation of ten measurements, the experimental conditions: 20 mM TMB, 0.2 mM H2O2, 0.2 mM acetate buffer (pH 4.0). (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)

size of GQDs which have a more intact aromatic structure and a rich periphery of carboxylic groups and unpaired electrons on its edge. ZnFe2O4/GQDs was also shown to be stable over a broad range of pH ranging from 2 to 12 and temperatures from 4 to 80 C (curve a in Fig. 2C and D). Conversely, the HRP lost most of its activity at pH lower than 3.0 and temperatures higher than 60 C (curve b in Fig. 2C and D). Thus, the robust nature of the ZnFe2O4/GQDs nanohybrid makes it more promising in a broad range of applications [26]. In addition, the peroxidase-like catalytic activities of the GS and Pd NWs were investigated as controls in Fig. S2 in Supporting information. 3.3. Characterization of the GS and Pd NWs/GS To develop a high-performance electrochemical DNA sensor, the critical issue was to enhance the immobilized amount of the capture probes on the electrode surface, and provide a good pathway for electron transfer. In this contribution, we tried to construct an improved DNA sensing interface using Pd NWs/GS for the immobilization of capture probes. Scanning electron microscopy (SEM) was used to characterize the stepwise fabrication process of the electrode. As can be seen in Fig. 3A, large GS was exfoliated, curled and resembled silk veil waves and exhibited the typical wrinkle morphology. In the inset of Fig. 3B, it can be seen that the Pd NWs had uniform shape and size. The average diameter was 5.0 nm along the entire length, which can be up to 1.5–2 mm. When Pd NWs were cast on GS, the GS were hidden behind the Pd NWs and was not so clear. Pd NWs were uniform in distribution on the surface of GS as shown in Fig. 3B.

3.4. Electrochemical impedance spectroscopy (EIS) of the immunosensor Electrochemical impedance spectroscopy (EIS) was used to monitor the interfacial properties of surface-modified electrode. There was a semicircle portion and a linear portion. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance. Fig. 3C shows the EIS upon the stepwise modification process in a solution of 5 mM [Fe(CN)6]3/4 and 0.1 M KCl. Curve a shows the EIS of the bare GCE, where a small semicircle was observed. When GS (curve b) and Pd NWs (curve c) were immobilized on the surface of GCE, the resistance was decreased accordingly, which was a strong proof that the GS and Pd NWs were an excellent electric conductive material that accelerated the electron transfer. Subsequently, when the electrode was conjugated with S1, the resistance was increased (curve d), because S1 blocked the electron exchange between the redox probe and the electrode, which suggested that the S1 were successfully immobilized on the surface. After the incubation of MCH (curve e) and the hybridization with S2 (curve f), the resistance increased again, which were caused by the nonconductive properties of biomacromolecule. Besides, the result suggested that the immobilized S2 on the electrode could retain their native bioactivity, and applied for the determination of it. 3.5. Cyclic voltammetry characterization Fig. 3D shows the cyclic voltammograms of the as-prepared MCH/S1/Pd/GS/GCE before and after hybridization with S2 and excess S3-GQDs/ZnFe2O4 in pH 7.4 Tris–HCl solution containing 0.1 mM thionine and 0.2 mM H2O2, respectively. A pair of stable and



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Fig. 3. SEM image of GS (A), Pd NWs/GS (B), the inset is the SEM image of Pd NWs. EIS of the bare GCE (a), GS/GCE (b), Pd NWs/GS/GCE (c), S1/Pd NWs/GS/GCE (d), MCH/S1/Pd NWs/GS/GCE (e), S2/MCH/S1/Pd NWs/GS/GCE (f) in 5.0 mM [Fe(CN)6]3/4 containing 0.1 M KCl (C). Cyclic voltammograms responses of different modified electrodes in pH 7.4 Tris–HCl solution containing 0.1 mM thionine and 0.2 mM H2O2: MCH/S1/Pd NWs/GS/GCE (a), S2/MCH/S1/Pd NWs/GS/GCE (b), ZnFe2O4/GQDs/S3/S2/ MCH/S1/Pd NWs/GS/GCE (c) (D).

well-defined redox peaks appeared at MCH/S1/Pd NWs/GS/GCE (curve a). After hybridization with S2, the currents peak decreased (curve b), indicated the achievement of the hybridization reaction on the surface of modified GCE that hindered the electron transfer. Upon hybridization with S3-GQDs/ZnFe2O4, an obvious catalytic process with the decrease of anodic peak and the increase of cathodic peak occurred (curve c). The catalytic current mainly attributed to the much higher enzyme activity of ZnFe2O4/GQDs, which reduced H2O2

with the aid of the TH as a mediator. Moreover, ZnFe2O4/GQDs could effectively shuttle electrons from the base electrode surface to the redox center of themselves. 3.6. Sensitive detection of target DNA Under the optimized experimental conditions (see in Supporting information), various concentrations of S2 were used to

Fig. 4. (A) DPV responses of the DNA biosensor in the presence (a–i) of different concentrations of S2 in pH 7.4 Tris–HCl solution containing 0.1 mM TH and 0.2 mM H2O2, S2 concentration (a) 0 (b) 1016 (c) 5 1015 (d) 1015 (e) 1014 (f) 1013 (g) 1012 (h) 1011 (i) 1010 (j) 5 1010 (k) 109 M. (B) The calibration curve of the DNA biosensor, each point was the average of ten measurements. (C) The interfering effects of sample matrix components on the current responses of the electrochemical DNA sensor.




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Table 1 Comparison between the proposed peroxidase mimic and other reported artificial enzymes for the determination of DNA hybridization. Peroxidase mimic

Linear range (nM)

LOD (nM)

Refs.

Hemin–graphene hybrid nanosheets Graphene Graphene-supported ferric porphyrin Single-walled carbon nanotube ZnFe2O4–graphene quantum dots

5–100 0–1000 1 107–0.01 – 1 107–5

2 11 2.2 108 1 6.2 108

[27] [28] [29] [30] This work

50 days. The good stability might be due to the good biocompatibility of Pd NWs/GS and ZnFe2O4/GQDs since they could retain the bioactivity of DNA. In addition, compared with the traditional enzyme labels, the enzyme-like ZnFe2O4/GQDs as inorganic materials were expected to be more stable and have higher catalytic activity than the natural enzymes.

investigate the sensitivity of the biosensor in pH 7.4 Tris–HCl solution containing 0.2 mM H2O2 and 0.1 mM TH by using ZnFe2O4/GQDs as trace and H2O2 as an enzyme substrate with the sensitive DPV detection. As shown in Fig. 4A and B, the reduction peak current increased gradually with the increase of the S2 concentration and the average reduction peak currents of the DNA biosensor were linearly proportional to the logarithm of S2 concentration in the range from 1016 to 5 109 M. The linear regression equation was I = 105.731 6.273 log c[S2] (R = 0.9993) with a detection limit (LOD) of 6.2 1017 M at a signal-to-noise ratio of 3s (where s is the standard deviation of the blank solution, n = 11). The low detection limit not only attributed to the enhanced catalytic performance of the ZnFe2O4/GQDs and the large specific surface area but good electrical conductivity of Pd NWs/GS. As shown in Table 1, our strategy was more sensitive than those of HRP and other mimic-based trace label as reported previously [27–30].

The practical application of the proposed mimic enzymatic electrochemical sensor was investigated by using standard addition method. Diluted human serum samples (human serum samples: buffer (1:10)) were used and several different concentrations of S2 were added as shown in Table 2. The recovery demonstrated the ability of the electrochemical biosensor for detection of DNA in human serum. The sensor would therefore offer an approach for accurate gene diagnostics.

3.7. Selectivity, stability and reproducibility of the immunosensor

4. Conclusion

The selectivity of the developed DNA sensor was assessed by using several hybridization experiments. For this purpose, the target DNA S2 and interfering strands S4 and S5 were used for hybridization, respectively. The DPV responses to each DNA strand and the mixed DNA strands are recorded in Fig. 4C. The signals from S4 and S5 solutions were no different from the blank, and the DPV responses of mixed DNA strands showed no obvious change compared with pure S2 which indicated that the sensor had an excellent selectivity to target the DNA and could be satisfactory to single-nucleotide polymorphism assays. The reproducibility and precision of the electrochemical DNA sensor was evaluated by relative standard deviation (RSD) of intra- and inter-assay. The intra- and inter-assay were evaluated by analyzing three levels of target DNA five times per run in 5 h. Experimental results indicated that the RSD of the intra-assay were 3.2, 5.8, and 3.9% at 1016, 1014 and 1012 M, respectively. Similarly, the inter-assay RSD of the DNA sensors made at the same electrode independently were 5.4%, 4.2%, and 3.4%. The low RSD indicated that the precision and reproducibility of the electrochemical DNA sensors were acceptable. The stability was a vital parameter in the performance of the prepared DNA biosensor. To investigate the stability, the DNA biosensor and bio-nanolabels were stored in pH 7.4 Tris–HCl at 4 C when not in use. No obvious change was observed after storage for 30 days but 8.6% decrease of the initial response was noticed after

This work demonstrated a novel peroxidase mimic ZnFe2O4/GQDs which was developed by loading GQDs onto ZnFe2O4. The ZnFe2O4/GQDs possesses higher peroxidase-like activity than each individual and HRP which was contributed to the unique properties of GQDs and the synergistic effect of the GQDs and ZnFe2O4. The ZnFe2O4/GQDs were also shown to be more stable over a broad range of pH and temperature range than HRP. Using the ZnFe2O4/GQDs as a trace, a novel electrochemical DNA sensor was developed by combining it with the Pd NWs/GS conductive materials modified electrode. The sensor exhibited much lower limit than that obtained with HRP and other mimic-based trace label. This work demonstrated the design of a novel HRP mimic with high peroxidase activity and the first use of the ZnFe2O4/GQDs mimic as a trace label for biosensing.

Table 2 DNA determination in serums by using the mimic enzymatic electrochemical sensor. Samples

1

2

3

4

5

Spiked/pM Proposed DNA sensora /pM Recovery/% RSD/%

0.005 0.0051 98 5.24

0.01 0.0096 96 2.83

0.1 0.098 98 3.63

10 10.3 103 3.15

100 101 101 2.51

a

The average value of ten successive determinations.

3.8. Application of the DNA biosensor in human serum samples

Acknowledgments This work was financially supported by National Natural Science Foundation of China (21277058, 51273084, 21175058); Natural Science Foundation of Shandong Province, China (ZR2012BZ002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.08.054. References [1] J. Huang, Y.R. Wu, Y. Chen, Z. Zhu, X.H. Yang, K.M. Wang, W.H. Tan, Pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids, Angew. Chem. Int. Ed. 50 (2011) 401–404. [2] L.G. Xu, Y.Y. Zhu, W. Ma, H. Kuang, L.Q. Liu, L.B. Wang, C.L. Xu, Sensitive and specific DNA detection based on nicking endonuclease-assisted fluorescence resonance energy transfer amplification, J. Phys. Chem. C 115 (2011) 16315–16321.



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