A sensitive electrochemical aptasensor for highly specific detection of streptomycin based on the porous carbon nanorods and multifunctional graphene nanocomposites for signal amplification

Sensors and Actuators B 241 (2017) 151–159

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A sensitive electrochemical aptasensor for highly specific detection of streptomycin based on the porous carbon nanorods and multifunctional graphene nanocomposites for signal amplification Junling Yin a , Wenjuan Guo a,∗ , Xiaoli Qin a , Juan Zhao a , Meishan Pei a , Feng Ding b,∗ a b

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Department of General Surgery, Jinan Hospital, No.63-1, Lishan Road, Jinan, Shandong Province 250013, China

a r t i c l e

i n f o

Article history: Received 1 May 2016 Received in revised form 9 October 2016 Accepted 12 October 2016 Available online 17 October 2016 Keywords: Electrochemical aptasensor Porous carbon nanorods Ferriferrous oxide nanoparticles Graphene Streptomycin

a b s t r a c t Quantitative detection of antibiotic residues in animal food stuffs is of great significance. In this work, a highly sensitive electrochemical aptasensor for the sensitive detection of streptomycin antibiotic was fabricated based on a novel signal amplification strategy. Specifically, this aptasensor was constructed utilizing porous carbon nanorods (PCNR) formed by porous carbon nanosphere and multifunctional graphene composite (GR–Fe3 O4 –AuNPs) as biosensing substrate. PCNR samples with large specific pore volume and high specific surface area were successfully prepared by hydrothermal and chemical activation treatment for the first time. GR–Fe3 O4 –AuNPs was served as labels to achieve a high sensitivity and low limit of detection (LOD). Under the optimized conditions, the proposed aptasensor exhibited a high sensitivity and a wider linearity to streptomycin in the range 0.05–200 ng/mL with a low detection limit of 0.028 ng/mL. The proposed aptasensor displayed an excellent analytical performance with great reproducibility, high selectivity and stability. In addition, the as–prepared aptasensor was successfully utilized for the determination of streptomycin in real samples. © 2016 Published by Elsevier B.V.

1. Introduction Streptomycin is an aminoglycoside antibiotic which is produced by Streptomyces griseus [1] and has been widely utilized in veterinary and human for treatment of gram-negative infectious disease [2,3]. Incorrect and uncontrolled application of streptomycin could result in the presence of antibiotic in foodstuffs and serious side effects on human health, such as nephrotoxicity and ototoxicity [4]. To date, various methods and strategies have been applied for the quantitative detection of streptomycin. Microbial inhibition assay, enzyme immunoassay and enzyme linked immuno-sorbent assay (ELISA) are commonly employed as screening tests but have cross-reactions with other substances in biological sample analysis [5]. Liquid chromatography-mass spectrometry (LC–MS) [6] and high performance liquid chromatography (HPLC) [7,8] have been conventionally described for confirmatory analysis. Although the chromatographic techniques are sensitive and specific, they are restricted to confirmatory analysis being very laborious and expen-

∗ Corresponding authors. E-mail addresses: chm [email protected] (W. Guo), [email protected] (F. Ding). http://dx.doi.org/10.1016/j.snb.2016.10.062 0925-4005/© 2016 Published by Elsevier B.V.

sive [9]. The sensitive determination of streptomycin with low cost is still a challenge in the practical applications. Therefore, it is highly desired to develop anaccurate and sensitive appraisal system to track the residual streptomycin. Aptamers are short single-stranded DNA (ssDNA) or RNA molecules, obtained by an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX) [10–12]. In addition, aptamers are able to specifically and selectively bind to their targets, ranging from small molecules to proteins and even cells [13,14]. Compared with traditional antibodies, aptamers possess the intrinsic advantages of its low cost, ease of synthesis and modification, excellent thermal stability and lack of immunogenicity and toxicity [15–17]. Owing to these advantages, numerous electrochemical aptasensors have been proposed and applied in the fields of food safety and clinical diagnosis. However, the reported aptamers specific to streptomycin are still very limited. In this work, an ssDNA aptamer [18] that binds to streptomycin with high affinity are introduced. In addition, the signal amplification is a key factor for the fabrication of aptasensors. Recently, enormous efforts have been devoted to the development of porous materials for signal amplification. As one kind of novel carbon material, ordered mesoporous carbon (OMC), has been receiving much attention in both scientific

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researches and practical applications owing to the extremely wellordered pore structure, high specific pore volume, high specific surface area, and tunable pore diameters in the mesopore range. Besides, high thermal stability and chemical inertness make them suitable for applications in sensing, catalysis, bioreactor construction, energystorage and capacitors, etc. [19]. Traditionally, porous carbon is prepared with SBA–15 mesoporous silicates as template, which is complicated and high cost [20]. However, in this paper, we conveniently prepared porous carbon nanosphere by the chemical activation with ZnCl2 . Interestingly, some nanospheres are fused together to form a pearl necklace and such structures could be described as “porous carbon nanorods (PCNR)” made of porous carbon nanospheres. This phenomenon should be due to the presence of [Fe(NH4 )2 (SO4 )2 ], which leads to the formation of rod–like carbon nanostructures instead of spheresby catalyzed hydrothermal heating in a sealed vessel [21]. Contrary to most poroussilica–based materials (for example, SBA–15) that are electronic semiconductors, the mesoporous carbons are intrinsical conductors [20].The high electrocatalytic activity observed at PCNR may attributed to the presence of a large number of edge plane graphite sites within the rod, since researchers have demonstrated that electrochemical reactions may proceed on carbon with spatial non–uniformity, and edge plane graphite sites/defects may generally show much more reactive than those at the basal–plane graphite toward electron transfer [22–24]. Hence, PCNR may have more interests and potential advantages for many advanced applications than other porous materials. Despite such potential capability of PCNR, there have been no studies on the electroanalytical applications for aptasensor. Currently, different signal amplification strategies have been created to improve the sensitivity and decrease the limit of detection (LOD) of the aptasensor. As a result, a wide variety of multifunctional nanomaterials have been designed as labels for different signal amplification strategies [25,26]. GR, a single–atom–thick sheet of sp2 –bonded carbon atoms, is often used as a substrate and GR-based composite materials have received increasing attention due to the synergistic contribution of two or more functional components and their potential applications [27]. Recently, metal and metal oxide nanoparticles have been widely applied to fabricate nanocomposites due to large surface-tovolume ratio, great electrical properties, strong adsorption ability, high surface reaction activity, small particle size and great surface properties [28], which are helpful for the immobilization of biomolecules.A series of nanomaterials based on Fe3 O4 NPs have been designed for signal amplification strategy because of its great biocompatibility and electrocatalytic properties toward the reduction of hydrogenperoxide (H2 O2 ) [29].In addition, gold nanoparticles (AuNPs) have been widely used in many applications because of their unique optical, physical and chemical properties [30–33]. What s more, AuNPs have good conductivity and biocompatibility and they can also form covalent bonds and combine with materials containing many functional groups, such as CN, NH3, or SH [34]. Thus, in this work, novel multifunctional grapheme nanocomposites (GR–Fe3 O4 –AuNPs) were constructed to achieve dual signal amplification strategy for the fabrication of aptasensor. In addition, the excellent performance of GR–Fe3 O4 –AuNPs is mainly due to these reasons: (1) Graphene was introduced to combine with Fe3 O4 NPs by chemical reaction and the obtained magnetic graphene nanocomposites (MGN) have a better electron transfer capability; (2) AuNPs were employed to functionalize the MGN to produce synergetic effect, which could result in the increasing of electrotransfer properties of the nanocomposites; (3) The introduction of AuNPs was beneficial to promote the biocompatibility of nanomaterials and increase the conjunction with aptamer. In present work, a novel electrochemical aptasensor based on PCNR/GR–Fe3 O4 –AuNPs was constructed for sensitive detection of streptomycin. Compared with the predecessors report, the pre-

pared aptasensor offered several advantages: (1) It was facile for aptamers to convert streptomycin into physically detect able electrochemical signals with high affinity and specificity; (2) PCNR with high electrocatalytic activity was synthesized simply and firstly applied for electroanalytical aptasensor; (3) The introduction of PCNR and GR nanocomposites greatly reduced the cost of the aptasensor; (4) Aptamers was immobilized on the electrode by Au-SH covalent bond rather than ␲-␲ weak interaction between aptamer and the surface of GR, which makes the aptasensor more stable. In addition, under the optimum conditions, the prepared aptasensor had a wider linear response range and a lower detection limit, which proved that the proposed aptasensor is sensitive and highly specific. More importantly, the as–prepared aptasensor could be used to determine streptomycin in milk. Thus, it may have potential applications for the detection of residual streptomycin in the field of food analysis. 2. Materials and methods 2.1. Reagents and materials The streptomycin aptamer (Apt), 5 -TAG GGA ATT CGT CGA CGG ATC CGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCC G-Thiol-3 was synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Ferric chloride (FeCl3 ·6H2 O) was purchased from Damao Chemical Reagent Tianjin Co., Ltd., China. Chloroauric acid (HAuCl4 ·3H2 O) was purchased from Sinopharm Chemical Reagent Shanghai Co., Ltd., China. Chitosan (CS), bovine serum albumin (BSA), glucose, HCl, trisodium citrate, ferrous ammonium sulfate hexahydrate (FeSO4 (NH4 )2 SO4 ·6H2 O) and zinc chloride (ZnCl2 ) were purchased from Aladdin Chemical Reagent Co., Ltd. (Beijing, China). All other chemicals were of analytical grade and received from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Distilled water was used throughout the experiment. 2.2. Apparatus Electrochemical experiments of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were carried out with a CHI 760E electrochemical workstation (Chenhua Instruments Co., Shanghai, China). Electrochemical impedance measurement (EIS) was performed with a Zennium electrochemical workstation (Zahner, Germany). Powder X–ray diffraction (XRD) data was obtained on a Bruker D8 advanced X–ray diffractometer using Cu K␣ radiation at a scan of 0.02◦ /s.The morphologies and energy-dispersive X–ray spectroscopy (EDS) of the samples were characterized by a QUANTA PEG 250 field emissionscanning electron microscope (SEM). N2 adsorption–desorption isotherms was carried out at 196 ◦ C using a micromeritics ASAP 2020 analyzer. Before adsorption, the samples were out-gassed at 120 ◦ C for 12 h. The specific surface area (SBET ) was evaluated using the Brunauer–Emmett–Teller (BET) method, and the mesopore volume was calculated according to the Barrett–Joyner–Halenda (BJH) formula and t–plot method, respectively. 2.3. Synthesis of PCNR PCNR was synthesized with glucose as carbon source by an improved and controllable hydrothermal synthetic route [21,35]. Typically, 4.0 g of glucose and 2.4 g FeSO4 (NH4 )2 SO4 ·6H2 O were dissolved in 40 mL of distilled water to form a clear solution, and then the solution was transferred into a Teflon–sealed autoclave and maintained at 180 ◦ C for 10 h. Subsequently, the products were impregnated in ZnCl2 (0.25 M) solution for 6 h. Finally, the material

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was activated in a N2 atmosphere at 300 ◦ C and the activated samples were thoroughly washed with distilled water and HCl solution (0.5 M).

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−0.2 to 0.6 V with modulation amplitude of 0.05 V, a pulse width of 0.05 s, and sample width of 0.0167 s. 3. Results and discussion

2.4. Preparation of GR-Fe3 O4 -AuNPs 3.1. Characterization of the prepared PCNR Graphene oxide (GO) was synthesized by an improved Hummers method [36]. In brief, a mixture of concentrated H2 SO4 (36 mL) and H3 PO4 (4 mL) was added to a mixture of graphite flakes (0.3 g) and KMnO4 (1.8 g), then the mixture was heated to 50 ◦ C and stirred for 12 h. After the system cooled to room temperature (RT), ice (40 mL) with 30% H2 O2 (0.3 mL) were poured. The mixture was centrifuged and the supernatant was decanted away. For workup, the remaining solid material was washed in succession with distilled water, 30% HCl, ethanol and ether. Finally the solid was dried in vacuum at 35 ◦ C. The powder was dispersed in distilled water by ultrasonication for 1 h and subsequently centrifuged for 15 min at 3000 rpm and dried overnight. GR–Fe3 O4 was prepared by the hydrothermal method [37]. Typically, FeCl3 ·6H2 O (1.0 g) was dissolved in ethylene glycol (20 mL) to form a clear solution, followed by the addition of sodium acetate (3.0 g), ethanediamine (10 mL) and a certain quality of GO. The mixture was stirred vigorously for 30 min and then sealed in a Teflon–sealed autoclave. The autoclave was heated and maintained at 200 ◦ C for 8 h, and then cooled down to RT. Finally, the products were washed several times with distilled water, and were dried at 50 ◦ C under high vacuum overnight. AuNPs were synthesized by the classical Frens method [38]. Briefly, a solution of HAuCl4 (0.01 wt%, 100 mL) was heated to boiling, and then a solution of trisodium citrate (1 wt%, 1.5 mL) was added. The boiling solution turned a brilliant ruby–red in around 15 min, indicating the formation of AuNPs, and then it was cooled to RT. A mixture of GR–Fe3 O4 (20 mg) and the as-prepared AuNPs solution (40 mL) was shaked for 12 h. The final product was obtained by being washed several times and dried at 35 ◦ C under high vacuum overnight. 2.5. Construction of the aptasensor The schematic diagram of the streptomycin aptasensor was shown in Scheme 1. Briefly, the GCE was firstly polished with 0.3 and 0.05 ␮m alumina slurry successively, and then washed in ethanol and distilled water thoroughly, respectively. Then, 5 ␮L of PCNR suspension was dropped onto the surface of the GCE and dried directly in air. Next, 5 ␮L GR–Fe3 O4 –AuNPs suspension was added onto the PCNR modified electrode surface. After washing with the PBS, the modified electrode was immersed into aptamer solution (5 ␮mol L−1 ) overnight. Then the electrode was washed with PBS to remove the unbound aptamer. Subsequently, 10 ␮L of 1% BSA was added onto the modified electrodeand incubated at 4 ◦ C for 2 h to block nonspecific binding sites. After washed by PBS thoroughly several times, the modified electrode was incubated in 10 mL streptomycin solution with different concentrations for 2 h. The as–prepared electrode was stored in the refrigerator prior to use. 2.6. Electrochemical measurements All electrochemical measurements were performed using a conventional three electrode system where a GCE served as the working electrode, a platinum wire as the counter electrode, and a KCl saturated Ag/AgCl as the reference electrode. 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) and 0.2 M KCl mixture in PBS were exploited in the EIS measurement. DPV was recorded in 5 mM Fe(CN)6 3−/4− containing 0.2 M KCl within the potential range of

Scanning electron microscope (SEM) is used to reveal the morphology and structure of prepared products. As can be seen from Fig. 1A, PCNR formed by porous carbon nanospheres are found. It seems that some particles are fused together to form a pearl necklace–like structure, which is corresponded with the TEM (Fig. S1). In addition, the XRD pattern (Fig. 1B) exhibits one broad diffraction peak at 2␪ angle of 10–30◦ , which is attributed to diffraction peak of amorphous carbon [39]. Amorphous carbon contains two–dimensional graphite layers or three–dimensional graphite crystallite and there are a large number of irregular bonds on the edge of microcrystalline, which largely determines the electrical conductivity of PCNR. In order to confirm the pore structure ofthe synthetic material, the N2 adsorption–desorption isotherms and pore size distribution of PCNR were displayed in Fig. 1C and D, respectively. The isotherm displayed a combination of type I and IV shape with pronounced H4–type hysteresis loop and the distinct hysteresis loop is characteristic of mesoporous materials, according to the IUPAC classification. The pore size distribution curve also suggests that the material simultaneously contains microporous and mesopores which are predominant with a major pore diameter of 2.7 nm. What s more, nanocomposite displayed a specific surface area (SSA) of 561.138 m2 g1 , indicating its high quality. 3.2. Characterization of the prepared GR–Fe3 O4 –AuNPs SEM images of GR–Fe3 O4 and GR–Fe3 O4 –AuNPs are helpful to identify their successful synthesis by observing their morphologies. As seen from Fig. 2A that GR still presented restacked sheets structure and numbers of Fe3 O4 NPs with a uniform diameter of about 130 nm were loaded on the surface of GR. In Fig. 2B, lots of AuNPs were successfully linked on the surface of GR–Fe3 O4 to obtain GR–Fe3 O4 –AuNPs, which possessed the advantages of AuNPs, Fe3 O4 NPs and GR. These results were also confirmed by TEM (Fig. S2). In addition, the energy dispersive X–ray spectrometry (EDS) can further explained what kinds of elements are contained in these nanomaterials. As shown in Fig. 2C and D, C, O and Fe elements are distributed throughout the GR–Fe3 O4 and C, O, Fe and Au elements throughout GR–Fe3 O4 –AuNPs. 3.3. Electrochemical characterization of the aptasensor In our work, PCNR and the complex of GR–Fe3 O4 –AuNPs were used to modify the electrode. The main function of PCNR was for the signal amplification. The GR–Fe3 O4 –AuNPs complex not only improves the current signal but also serves for aptamers immobilization. Results showed that the introduction of Fe3 O4 enhanced current signal. The introduction of AuNPs was mainly for the immobilization of the streptomycin aptamers on the electrode via the strong bonding interaction between AuNPs and the thiol group. Although aptamer has been known to be absorbed on the surface of GO/graphene via ␲–␲ interaction, in this work, the introduction of numbers of Fe3 O4 NPs and AuNPs loaded on the surface of GR could reduce the ␲-␲ interaction between aptamer and the surface of GR. Thus, the aptamer has been loaded on the surface of the modified electrode mainly via the Au-SH covalent bonds rather than the ␲–␲ interaction. In addition, the strong interaction has been known to be superior to ␲-␲ weak interaction [36]. In order to characterize of the different modified electrodes furtherly, CVs and EIS has been carried out.

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Scheme 1. Schematic diagram of the streptomycin aptasensor.

Fig. 1. (A) SEM, (B) XRD, (C) N2 adsorption/desorption isotherms and (D) and BJH pore size distribution of PCNR.

All CVs of different modified electrodes were recorded from −0.2 to 0.6 V in 5 mM Fe(CN)6 3−/4− containing 0.2 M KCl at a scan rate of 100 mV/s. In Fig. 3A, it can be clearly seen that GR modified electrode showed a couple of better-defined redox peaks (curve b) compared with GCE (curve a), mainly owing tothe excellent electron transfer of GR. The peak currents of the GR–AuNPs/GCE (curve c) and GR–Fe3 O4 /GCE (curve d) were superior tothat of the GR/GCE, indicating that the GR–based composite materials could increase the current signal significantly. The peak

current of GR–Fe3 O4 –AuNPs/GCE (curve e) was larger than that of GR–AuNPs/GCE and GR–Fe3 O4 /GCE, ascribing the synergistic amplification effect of three kinds of nanomaterials. Additionally, the detection of streptomycin (120 ng/mL) was performed by GR–AuNPs and GR–Fe3 O4 –AuNPs as aptasensor platform, respectively. The result in inset curve of Fig. 3A also indicated the developed aptasensor platform GR–Fe3 O4 –AuNPs (curve b) was over GR–AuNPs (curve a).

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Fig. 2. SEM images of (A) GR–Fe3 O4 and (B) GR–Fe3 O4 –AuNPs; EDS of (C) GR–Fe3 O4 and (D) GR–Fe3 O4 –AuNPs.

As shown in Fig. 3B, a pair of well–defined reversible redox peaks was observed at the bare GCE (curve a) suggesting a reversible electrochemical process. After PCNR was immobilized on the electrode surface, the peak current (curve b) was increased significantly due to the excellent electrical conductivity and larger special surface area of the PCNR. Also the peak current of the GR–Fe3 O4 –AuNPs/PCNR/GCE (curve c) increased about 90 ␮A than that of the PCNR/GCE. This phenomenon should be attributed to the excellent electrical conductivity and the great electroactivity of GR–Fe3 O4 –AuNPs. After streptomycin aptamer was adsorbed onto the electrode surface, there was an obvious decrease of the peak current (curve d), indicating that the aptamer can generate the insulating layer and hinder electron transfer. When BSA was employed to block extra active sites and avoid the nonspecific adsorption, a successive decrease in the current (curve d) was observed. Subsequently, the reaction between the aptamer and streptomycin led to a further decrease of current signal (curve f), indicating that the streptomycin was successfully captured by the aptamer and the aptamer–streptomycin complex layer blocks the electron transfer. EIS is an effective method to further characterize the electron transfer properties of the different modified electrodes. The impedance spectrum consisted of a semicircle portion and a linear portion. Note that the diameters of the semicircles were equal to the charge–transfer resistance (Ret ), which controlled the electron transfer kinetics of the redox probe at the electrode interface. EIS was carried out in a background solution of 0.1 M PBS containing 5.0 mM Fe(CN)6 3−/4− and 0.2 M KCl in the frequency range of 0.1–105 Hz with an amplitude of 0.005 V. The Nyquist diagrams of

the modified electrodes at different stages and the corresponding equivalent circuit were displayed in Fig. 3C. It is observed that a relatively larger interface electron transfer resistance was obtained at the GCE (curve a). After modified with PCNR, a smaller resistance (curve b) was observed attributing tothe good conductivity of PCNR. Compared with Rct of the PCNR/GCE, GR–Fe3 O4 –AuNPs/PCNR/GCE (curve c) modified electrode was further decreased, indicating that GR–Fe3 O4 –AuNPs can accelerate electron transfer and increase the electrode surface area. When aptamer was assembled on the GR–Fe3 O4 –AuNPs/PCNR/GCE, the Rct was increased significantly (curve d), suggesting that aptamer was immobilized on the electrode and blocked the electron exchange between the redox probe and electrode. Moreover, the capture of BSA (curve e) and streptomycin (curve f) resulted in the further increase of Rct , implying that electron transfer becomes more difficult. The electroactive surface areas (A) of the different kinds of electrodes were calculated based on the Randles–Sevcike quation Ip = 2.65 × 105 n3/2 AD1/2 1/2 C, where Ip is the peak current, n is the transferring electron number, A is the electroactive area (cm2 ), D is the diffusion coefficient,  is the scan rate, and C is the concentration of the substrate [40]. The diffusion coefficient of K3 [Fe(CN)6 ] is 7.6 × 106 cm2 /s [41]. The calculated results are listed in Table 1. Results showed that values of A were calculated to be 0.027 cm2 , 0.468 cm2 , 0.471 cm2 , and 0.513 cm2 for GCE, PCNR/GCE, GR–Fe3 O4 –AuNPs/GCE and GR–Fe3 O4 –AuNPs/PCNR/GCE, respectively. A of PCNR/GCE and GR–Fe3 O4 –AuNPs/GCE modified electrode is larger than that of GCE, which further proved that nano–materials possess excellent

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Fig. 3. (A) CVs of bare GCE (a), GR/GCE (b), GR–AuNPs/GCE (c), GR–Fe3 O4 /GCE (d) and GR–Fe3 O4 –AuNPs/GCE (e); the inset of A: DPVs of streptomycin/BSA/aptamer/GR–AuNPs/ PCNR/GCE (a), streptomycin/BSA/aptamer/GR–Fe3 O4 –AuNPs/PCNR/GCE (b); (B) CVs and (C) EIS of bare GCE (a), PCNR/GCE (b), GR–Fe3 O4 –AuNPs/PCNR/GCE (c), aptamer/GR–Fe3 O4 –AuNPs/PCNR/GCE (d), BSA/aptamer/GR–Fe3 O4 –AuNPs/PCNR/GCE (e), streptomycin/BSA/aptamer/GR–Fe3 O4 –AuNPs/PCNR/GCE (f), the inset of C: the equivalent circuit of the Nyquist plots.

Fig. 4. Influence of the pH and incubation time of streptomycin on the DPV peak current of the aptasensor.

Table 1 The electroactive surface area (A) of different modified electrodes. Electrode

A(cm2 )

GCE PCNR/GCE GR-Fe3 O4 -AuNPs/GCE GR-Fe3 O4 -AuNPs/PCNR/GCE

0.057 0.468 0.471 0.513

electrical conductivity. A of GR–Fe3 O4 –AuNPs/PCNR/GCE is larger than PCNR/GCE and GR–Fe3 O4 –AuNPs/GCE modified electrodes, which suggests the aptasensor successfully combine advantages of PCNR and GR–Fe3 O4 –AuNPs. 3.4. Optimization of experimental conditions In order to achieve an optimal electrochemical signal, the optimization of experimental conditions was necessary. The value of pH mainly influences the structure and performance of aptamer and the activity of biological materials. Fig. 4A shows the different electrocatalytic current responses of the aptasensor for the detection of 180 ng/mL of streptomycin in different pH values of PBS. As shown in Fig. 4A, the optimal amperometric response was achieved at pH = 7.4. Higher or lower pH resulted in a decrease of electrocatalytic current responses. The results indicated that strong acidic and alkaline solutions can damage the structure and performance of aptamer, and further reduce the affinity between aptamer and the electrode surface. Therefore, PBS at pH = 7.4 was selected for the test throughout this study. The incubation time is also an important parameter for the aptasensor to achieve maximized current signal. A series of modified electrodes were incubated with 180 ng/mL streptomycin for 20, 30, 50, 70, 90, 100, 120, 160 and 180 min, respectively. Fig. 4 B shows the curves corresponding to the DPV detection of strep-

Fig. 5. Calibration curve of DPV peak currents for different streptomycin concentrations from 0.05 to 200 ng/mL. The inset shows DPV responses of the electrochemical aptasensor to different concentrations of streptomycin (from a–j: 0, 0.05, 5, 25, 50, 100, 120, 150, 180, 200 ng/mL).

tomycin in 0.1 M PBS (pH = 7.4) based on the changes of current intensity (I) between before and after streptomycin incubation. I increases significantly with the increase of incubation time and tends to a steady value after 120 min, which suggests that the building of streptomycin-aptamer complex reaches the saturation. Therefore, 120 min was chosen as the optimized incubation time for the determination of streptomycin. 3.5. Sensitivity of the aptasensor Under optimal conditions, the modified electrodes were incubated in different concentrations of streptomycin. As shown in Fig. 5, I increases linearly with the increase of the concen-

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Table 2 Comparison of streptomycin determinations using the proposed and reference methods. Sensors

Linear range

Limit of detection

Reference

mMIP-based sensor Apt-CS-modified electrodes Colorimetric and fluorescence quenching LC–MS/MS FIA-EQCN GR-Fe3 O4 -AuNPs/PCNR/GCE

0.05–20 ng/mL 44–218 ng/mL 2.9–102 ␮g/mL 1.0–20 ng/mL 0.3–10 ng/mL 0.05–200 ng/mL

10 pg/mL 16.6 ng/mL 0.29 ␮g/mL 2.0 ng/mL 0.3 ng/mL 0.028 ng/mL

[42] [18] [43] [44] [45] Present work

Table 3 Streptomycin determination in milk by the proposed aptasensor (RSD: Rlative Sandard Deviation). Streptomycin

Electrochemical method

ng/mL

ng/mL ± RSD(%)

Recovery (%)

ng/mL

RSD (%)

Recovery (%)

RSD(%)

2.5 5.0 8.0 10.0

2.5 ± 1 5±2 8±5 10± 3

100 104 98 101

2.5 5 8 10

2 3 3 2

99 103 97 102

3 2 5 4

ELISA method

tration of streptomycin in the range of 0.05–200 ng/mL with a correlation coefficient of 0.9987. The regression equation was I (␮A) = 0.72c + 11.23(ng/mL). A detection limit of 0.028 ng/mL was achieved (S/N = 3). The proposed method forthe determination of streptomycin was compared with the previously reported methods in Table 2. The low limit of detection (LOD) can be considered as followed: (1) PCNR samples possess great electrical conductivity due to their pore structure, high specific surface area and the presence of a large number of edge plane graphite sites within the rod, which are in favour of electron transfer; (2) GR–Fe3 O4 –AuNPs combine the advantages of GR, Fe3 O4 NPs and AuNPs to achieve dual signal amplification strategy for the fabrication of aptasensor and promote the biocompatibility of nanomaterials with aptamer. From Table 2, we can see that compared with other methods, the as–proposed method has a relatively high sensitivity and low detection limit.

HPLC method

Fig. 6. DPV current responses of the aptasensor to (a) streptomycin (b–e) interferents, and (f–i) mixtures of streptomycin and different interferents. Error bars are standard deviations across three repetitive experiments.

3.6. The stability, specificity and reproducibility of the aptasensor Stability is a key parameter for developing a practical aptasensor. To evaluate the stability of the aptasensor, five electrodes were independently prepared and stored at 4 ◦ C before use. The current response for the aptasenor decreased 5% after two weeks storage, demonstrating that the proposed aptasensor exhibited a sufficient stability for the detection of streptomycin. Specificity is another important criterion for an electrochemical aptasensor. In our work, the current response of the prepared aptasensor to streptomycin (100 ng/mL), glucose, penicillin, ascorbic acid, methionine, mixtures of streptomycin (100 ng/mL) and interfering substances (10 ng/mL) were studied. As shown in Fig. 6, streptomycin showed a much stronger current response (Fig. 6a) while a weak current response was obtained in the presence of those interferents (Fig. 6b-e). In addition, mixtures of streptomycin and interferents also showed a much stronger current response (Fig. 6f-i). Based on above results, we can clearly observe that the as–prepared aptasensor had high selectivity for the detection of streptomycin. To evaluate the reproducibility of the aptasensor, a series of aptasensors fabricated on ten electrodes were prepared for the detection of 25 ng/mL streptomycin. The relative standard deviation (RSD) of the measurements was 3.1%, which indicates that the reproducibility of the aptasensorwas acceptable. 3.7. Determination of streptomycin in real samples In order to study the possibility and precision of the proposed aptasensor, it is worthy to demonstrate the feasibility of the pro-

posed aptasensor for practical application. Milk is usually used as one of the most important regulated products in food analysis owing to the risk of having veterinary medicine residue. Thus, it is very necessary to detect streptomycin in milk. In our work, the potential application of developed aptasensor hasbeen tested for the detection of SRT in the diluted milk samples. Initially, all the milk samples were assumed to be free of SRT. Streptomycin standard solutions were spiked into the 10 × diluted milk with PBS (pH = 7.4) to prepare the streptomycin concentrations of 2.5, 5.0, 8.0 and 10.0 ng/mL. Several aptasensors were used to detect the different concentrations of streptomycin. Based on the response signals recorded from proposed aptasensor, recoveries were calculated. As shown in Table 3, the recovery of streptomycin concentration ranges from 97.9% to 104.1%. To further investigate the practical application of the aptasensor, reference enzyme–linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) [46] methods were performed. These data obtained using our method was in good agreement with those obtained by utilizing ELISA and HPLC methods. Therefore, the constructed aptasensor could be effectively applied to the quantitative detection of streptomycin in real samples. 4. Conclusion In this work, a novel aptasensors based on PCNR and GR–Fe3 O4 –AuNPs nanocomposites for dual signal amplification for quantitative detection of streptomycin has been successfully constructed. PCNR and GR–Fe3 O4 –AuNPs nanocomposites served as matrix greatly reduced the aptasensor cost and the immobilization

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of aptamer by covalent bond enormously improved the stability of sensor. Compared to the previously reported literature, the signalattenuated electrochemical sensing developed in this paper was more sensitive. In addition, the constructed aptasensor offered the advantages of improved specificity, and reproducibility, and this analytical method will have promising applications both in the field of environmental analysis and food safety control. Of greater significance, this highly sensitive electrochemical aptasensor could be widely extended to the detection of other antibiotics by replacing the sequence of the aptamer. Acknowledgements This work was supported financially by Shandong Provincial Natural Science Foundation, China (Grant No. ZR2012BL11), and Shandong Provincial Science and Technology Development Plan Project, China (Grant No. 2013GGX10705). 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.snb.2016.10.062. References [1] M.H. Lan, W.M. Liu, J.C. Ge, J.S. Wu, J.Y. Sun, W.J. Zhang, P.F. Wang, A selective fluorescent and colorimetric dual-responses chemosensor forstreptomycin based on polythiophene derivative, Spectrochim. Acta Part A 136 (2015) 871–874. ˜ R.A.M. Zucchetti, R.E.M. Nino, ˜ R. Patel, A.G. [2] R.H.M.M. Granja, A.M.M. Nino, Salerno, Determination of streptomycin residues in honey by liquid chromatography–tandem mass spectrometry, Anal. Chim. Acta 637 (2009) 64–67. [3] N. Zhou, J. Wang, J. Zhang, C. Li, Y. Tian, J. Wang, Selection and identification of streptomycin-specific single-stranded DNA aptamers and the application in the detection of streptomycin in honey, Talanta 108 (2013) 109. [4] R.C.D. Oliveira1, J.A.R. Paschoal1, M. Sismotto1, F.P.D.S. Airoldi, F.G.R. Reyes, Development and validation of an LC-APCI-MS–MS analytical method for the determination of streptomycin and dihydrostreptomycin residues in milk, J. Chromatogr. Sci. 47 (2009) 756–761. [5] X.B. Feng, N. Gan, S.C. Lin, T.H. Li, Y.T. Cao, F.T. Hu, Q.L. Jiang, Y.J. Chen, Ratiometric electrochemiluminescent aptasensor array for antibiotic based on internal standard method and spatial-resolved technique, Sens. Actuators B 226 (2016) 305–311. [6] A.M. Gremilogianni, N.C. Megoulas, M.A. Koupparis, Hydrophilic interaction vs ion pair liquid chromatography for the determination of streptomycin and dihydrostreptomycin residues in milk based on mass spectrometric, J. Chromatogr. A 1217 (2010) 6646–6651. [7] M. Ramezani, N.M. Danesh, P. Lavaee, K. Abnous, S.M. Taghdisi, A selective and sensitive fluorescent aptasensor for detection of kanamycin based on catalytic recycling activity of exonuclease III and gold nanoparticles, Sens. Actuators B 222 (2016) 1–7. ˜ [8] P. Vinas, N. Balsalobre, C.M. Hernández, Liquid chromatography on an amide stationary phase with post-column derivatization and fluorimetric detection for the determination of streptomycin and dihydrostreptomycin in foods, Talanta 72 (2007) 808. [9] H.Y. Song, T.I. Wang, S.F. Guo, J. Ding, C. Tan, S. Gorelik, X.D. Zhou, Nanoimprinted thrombin aptasensor with picomolar sensitivity based on plasmon excited quantum dots, Sens. Actuators B 221 (2015) 207–216. [10] X. Tang, Y.S. Wang, J.H. Xue, B. Zhou, J.X. Cao, S.H. Chen, M.H. Li, X.F. Wang, Y.F. Zhu, Y.Q. Huang, A novel strategy for dual-channel detection of metallothioneins and mercury based on the conformational switching of functional chimera aptamer, J. Pharm. Biomed. Anal. 107 (2015) 258–264. [11] B. Zhao, P. Wu, H. Zhang, C. Cai, Designing activatable aptamer probes for simultaneous detection of multiple tumor-related proteins in living cancer cells, Biosens. Bioelectron. 68 (2015) 763–770. [12] R. Liu, Z.H. Yang, Q. Guo, J.C. Zhao, J. Ma, Q. Kang, Y.F. Tang, Y. Xue, M. He, Signaling-Probe displacement electrochemical aptamer-based sensor (SD-EAB) for detection of nanomolar kanamycin A, Electrochim. Acta 182 (2015) 516–523. [13] Y.S. Liu, J. Yu, Y. Wang, Z.Y. Liu, Z.S. Lu, An ultrasensitive aptasensor for detection of Ochratoxin A based on shielding effect-induced inhibition of fluorescence resonance energy transfer, Sens. Actuators B 222 (2016) 797–803. [14] P. Luo, Y. Liu, Y. Xia, H. Xu, G. Xie, Aptamer biosensor for sensitive detection of toxin A of Clostridium difficile using gold nanoparticles synthesized by Bacillus stearothermophilus, Biosens. Bioelectron. 54 (2014) 217–221.

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Biographies Junling Yin is currently a postgraduate student at the School of Chemistry and Chemical Engineering, University of Jinan, China. She obtained her Bachelor degree at the School of Chemistry and Chemical Engineering, University of Jinan in 2014. Her main research interests focus on electrochemical aptasensor, immunoassays and conducting polymers.

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Wenjuan Guo obtained her PhD degree at the department of Chemistry, Shandong University in 2007. Now she is working as a teacher at the department of Chemistry, University of Jinan, China. Her main research interests focus on electrochemical immunosensor, immunoassays and conducting polymers. Xiaoli Qin is currently a postgraduate student at the School of Chemistry and Chemical Engineering, University of Jinan, China. She obtained her Bachelor degree at the School of Chemistry and Chemical Engineering, University of Jinan in 2013. Her main research interests focus on electrochemical aptasensor, immunoassays and conducting polymers. Juan Zhao is currently a postgraduate student at the School of Chemistry and Chemical Engineering, University of Jinan, China. She obtained her Bachelor degree at the School of Chemistry and Chemical Engineering, University of Jinan in 2014. Her main research interests focus on electrochemical immunosensor, immunoassays and conducting polymers. Meishan Pei is currently a Full Professor at the department of Chemistry, University of Jinan, China. He obtained his PhD degree in Institute of chemistry, Chinese academy of sciences in 2004. His research focuses on the synthesis and application of concrete admixtures, conductive polymers, Oilfield chemicals and electrochemical immunosensor, immunoassays. Feng Ding is currently a doctor at the Department of General Surgery, Jinan Hospital.