Biosensors and Bioelectronics 74 (2015) 691–697
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A novel electrochemical aptasensor for ultrasensitive detection of kanamycin based on MWCNTs–HMIMPF6 and nanoporous PtTi alloy Wenjuan Guo a, Na Sun b, Xiaoli Qin a, Meishan Pei a,n, Luyan Wang a a Shandong Provincial Key Laboratory of Chemical Sensing & Analysis, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China b Environmental Protection Monitoring Station, Jining 272045, China
art ic l e i nf o Article history: Received 16 April 2015 Received in revised form 22 June 2015 Accepted 23 June 2015 Available online 10 July 2015 Keywords: Aptasensor Ionic liquid Nanoporous Platinum Kanamycin
a b s t r a c t A novel aptasensor based on a novel composite film consisting of multi-walled carbon nanotubes (MWCNTs), ionic liquid (IL) of 1-hexyl-3-methylimidazolium hexafluorophosphate (HMIMPF6), and nanoporous PtTi (NPPtTi) alloy was constructed for ultrasensitive detection of kanamycin. The NP-PtTi alloy was successfully fabricated by a simple dealloying of PtTiAl source alloy in HCl solution. The NP-PtTi alloy has uniform interconnected network structure with specific surface area and was used to immobilize aptamer. After modified with the composite material, current signal was amplified obviously, which attributed to the larger specific surface area and excellent electrical conductivity of NP-PtTi and MWCNTs. A number of factors affecting the activity of the aptasensor have been discussed and optimized. Under the optimized conditions, the proposed aptasensor provided a linear range of 0.05–100 ng mL 1 with a low detection limit of 3.7 pg mL 1. This aptasensor displayed high sensitivity, stability and reproducibility. In addition, the as-prepared aptasensor was successfully used for the determination of kanamycin in a real sample. & 2015 Published by Elsevier B.V.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Reagents and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fabrication of NP-PtTi alloy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Construction of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Electrochemical measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Characterization of the prepared NP-PtTi alloy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SEM characterization of modified electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Electrochemical characterization of the modified electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Optimization of experimental conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Sensitivity of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. The stability, specificity and reproducibility of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Determination of kanamycin in real samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author. Fax: þ 86 531 82765475. E-mail address: [email protected] (M. Pei).
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1. Introduction Kanamycin is an aminoglycoside antibiotic which is produced by the fermentation of streptomyces kanamyceticus (Spahn and Prescott, 1996; Bai et al., 2014). It is used to treat a variety of infections by inducing mistranslation and indirectly inhibiting translocation during protein synthesis (Wirmer and Westhof 2006; Song et al., 2011). The abuse of kanamycin can cause serious side effects, such as loss of hearing, toxicity to the kidneys, and allergic reactions to the drugs (Bai et al., 2014; Wirmer and Westhof 2006). To protect the customers’ security, the European Union (EU) has established maximum residue limits (MRLs) for kanamycin in food, such as 100 μg kg 1 for meat, 600 μg kg 1 for liver, 2500 μg kg 1 for kidney, and 150 μg kg 1 for milk ( Scheme 1). At present, many analytical methods have been reported for the detection of kanamycin. A label-free immunosensor was constructed based on nanoparticles and graphene sheet mixed with thionine (Yu et al., 2013). An immunosensor was fabricated based on water-soluble graphene sheet/prussian blue-chitosan/nanoporous gold composited film modified electrode (Zhao et al., 2011). Chen et al. (2013) prepared an immunosensor based on MRS assay and bovine serum albumin (BSA) system. An electrochemical aptasensor based on the synergistic contributions of multiple nanocomposites for kanamycin detection has been reported (Sun et al., 2014). The constructing process of the sensors motioned above usually involved complicated assembly processes, and the usage of a large amount of reagents. The quick determination of kanamycin with low cost is still a challenge in the practical applications. It is essential to develop the applicable techniques for simple, low-cost and sensitive detections. Aptamers, the synthetic single-stranded DNA or RNA molecules with specific three-dimensional (3D) structures, are selected in vitro through systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, 1990; Zhu et al., 2012). Aptamers can be used as the recognition probes superior to antibodies because of their advantages such as good stability, easy modification, high affinity and specificity (Wei et al., 2012). Thus, the electrochemical aptasensors are especially attractive in the fields of food safety and clinical diagnosis. Recently, aptasensors (Zhu et al., 2012; W. Xu et al., 2014) have been used in many field, such as chemiluminescence (Yan et al., 2012; Yu et al., 2011a, 2011b), electrochemistry (Sun et al., 2014), colorimetric (Wang et al., 2012), and immunoassay (Ge et al., 2012a, 2012b; Zang et al.,
2012). Among them, the electrochemical detection based on aptasensor has shown significant advantages, such as high sensitivity, fast response time, simple operation, and low cost. The signal amplification is a key factor for the construction of aptasensor. Strategies based on nanomaterials have promising potential in ultrasensitive biological detection due to the versatile properties of nanomaterials. Various nanomaterials including Au, Ag, Cu, Pd, and Pt-based metals have been used in constructing biosensors with high sensitivity (Wang et al., 2014; Liu et al., 2013; Chen et al., 2012). At present, the nanoporous materials are particularly attractive in bioassays due to their large specific surface area. Nanoporous Pt-based alloy exhibited more active sites than the single Pt, Pd, and Au nanoparticles (Crumbliss et al., 1992; Xu et al., 2010). PtTi alloy nanoparticles of various ratios were prepared by using Pt2(dba)3 and titanium chloride (TiCl4) as precursors (Ding et al., 2008). Results showed that PtTi/C alloy nanoparticles showed higher activity than Pt/C catalyst. PtTi-based ternary alloy was found to exhibit higher activity than pure Pt electrocatalyst (He and Kreidler, 2008). A nanoporous PtTi alloy with multimodal ligament/pore structure was designed, which showed high activity and high catalytic durability (Duan et al., 2015). Based on that point, in this paper, nanoporous Pt–Ti (NPPtTi) alloy with uniform nanoporous structure was successfully fabricated by selectively dealloying Al from a PtTiAl alloy in HCl solution. NP-PtTi alloy with excellent electrical conductivity has three-dimensional (3D) interconnected network structure, which increases its large specific surface area and provides a beneficial immobilization platform for kanamycin aptamer carrier. Till now, carbon nanotubes (CNTs) have gained increasing interests in a great variety of research fields (Gopalan et al., 2009; Tunckol et al., 2013; Liu et al., 2011) due to their high electrical conductivity, chemical stability, and remarkable mechanical strength. Even though MWCNTs were hydrophobic and difficult to be dispersed by ordinary solvents, many approaches have been developed to overcome the above obstacle, including sidewall functionalization (Holzinger et al., 2003), polymer wrapping (Zheng et al., 2003), and addition of surfactants (Moore et al., 2003). However, these methods did not avoid some drawbacks, such as time-consuming, complicated physical and chemical reaction. With the development of IL, it is found that CNTs could be easily untangled into much finer bundles in the IL (Xiao et al., 2008). Aida’s group (Fukushima et al., 2003) prepared a gel containing pristine single-walled carbon nanotubes mixed with IL to
Scheme 1. Schematic diagram of the kanamycin aptasensor.
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improve the performance of electrodes. Our group (Guo et al., 2014) also found the synergic effect of the MWCNTs and IL. Results showed that MWCNTs–IL composite could be used as an effective load matrix and the enhancement effect of the current response is significant, which provided a new way for constructing the powerful biosensors with excellent properties. In this paper, a novel electrochemical sensor for ultrasensitive detection of kanamycin was fabricated based on MWCNTs–1-hexyl3-methylimidazolium hexafluorophosphate (HMIMPF6) (MWCNTs– HMIMPF6) and NP-PtTi alloy as the matrix. MWCNTs–HMIMPF6/NPPtTi alloy nanocomposite constructed an effective biomolecules immobilization matrix with unblocked conductive pathway for electron transfer. More importantly, MWCNTs–HMIMPF6/NP-PtTi nanocomposite was first used in electrochemical sensors as well with significant signal improvement. Under the optimum conditions, the prepared aptasensor had a wide linear response range and a lower detection limit, which proved this method to be an efficient method of constructing electrochemical aptasensor with highly sensitivity and specificity for the quantitative detection of kanamycin. In addition, the results from the electrochemical measurement were compared with that from ELISA method (W. Xu et al., 2014; Chen et al., 2013) for determining kanamycin in milk, which proved the as-prepared aptasensor was a useful tool for the detection of kanamycin in real samples.
2. Materials and methods 2.1. Reagents and materials A kanamycin aptamer modified with an amino group at the 5′ position, namely 5′-NH2-AGATGGGGGTTGAGGCTAAGCCGA-3′ was synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Kanamycin aptamer was selected by systematic evolution of ligands by exponential enrichment (SELEX) that could specifically bind with kanamycin (Zhu et al., 2012; W. Xu et al., 2014). MWCNTs were obtained from Beijing Dekedaojin technology Co., Ltd. (China). HMIMPF6, chitosan (CS), bovine serum albumin (BSA), glucose, ascorbic acid, uric acid, methionine, and kanamycin 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). Ultrapure water was used throughout the experiment. The concentration of kanamycin obtained based on the enzyme-linked immunosorbent assay (ELISA) method was provided by the Aladdin Chemical Reagent Co., Ltd. and the previous papers (Zhu et al., 2012; Sun et al., 2014). ELISA is a popular format of analytic biochemistry assay that uses an antibody to detect the presence of an antigen. The basic principle of an ELISA is to detect signal produced by enzyme which is linked to the antibody. 2.2. Apparatus Electrochemical experiments of CV and DPV were carried out with a CHI 760E electrochemical workstation (Chenhua Instruments Co., Shanghai, China). EIS was performed with a Zennium electrochemical workstation (Zahner, Germany). The morphologies and energy-dispersive X-ray spectroscopy (EDS) of the samples were characterized by a QUANTA PEG 250 field emission scanning electron microscope (SEM). Transmission electron microscopy (TEM) was obtained with a JEM-2100 high resolution transmission electron microscope. Powder X-ray diffraction (XRD) data were obtained on a Bruker D8 advanced X-ray diffractometer using Cu Kα radiation at a scan of 0.04°/s. Grain size distribution of NP-PtTi alloy was tested by a laser particle size analyzer (Britain, Malvern).
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2.3. Fabrication of NP-PtTi alloy Pt10Ti10Al80 ternary alloy foils were prepared by refining the corresponding metals Pt, Ti, and Al (99.99%) under an Ar-protected atmosphere in an arc-furnace, followed by melt-spinning (Duan et al., 2015; C.X. Xu et al., 2014; Zhang et al., 2009). Pt10Ti10Al80 alloy foils were dealloyed in 1 M HCl solution at room temperature (RT) for 30 min to obtain NP-PtTi alloy by etching the Al atoms and part of Ti atoms. After dealloying, the product was washed several times with ultrapure water and dried at RT in air. 2 mg NP-PtTi alloys were dispersed in 1 mL of 1 wt% CS under sonication to obtain a homogeneous suspension. 2.4. Construction of the aptasensor The bared GCE was polished with 0.3 and 0.05 μm alumina slurry, and then thoroughly washed in ethanol and ultrapure water, respectively. MWCNTs–HMIMPF6 composite was fabricated according to the literature (Guo et al., 2014). 5 μL MWCNTs–HMIMPF6 suspensions were dropped onto the surface of the GCE, and then dried directly in air. Next, 5 μL NP-PtTi suspensions were dropped onto the MWCNTs–HMIMPF6 modified electrode surface. After washing with the PBS (pH ¼ 7.4), the modified electrode was immersed into 10 μL of 5 μmol L 1 aptamer solution overnight. Then the electrode was washed with PBS to remove the unbound aptamer. 10 μL of 1% BSA solution were employed to block nonspecific binding sites for 2 h at 4 °C. After washed by PBS thoroughly several times, the modified electrode was incubated in 10 mL kanamycin solution with different concentrations for 2 h. The as-prepared electrode was stored in the refrigerator prior to use. The schematic diagram of the kanamycin aptasensor is shown in Fig. 1. 2.5. Electrochemical measurements All electrochemical measurements were performed using a conventional three-electrode system composed of a GCE as the working electrode, a platinum wire as the counter electrode, and a KCl saturated Ag/AgCl as the reference electrode. The EIS measurement was performed in the presence of 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) and 0.2 M KCl mixture in PBS (pH¼7.4). DPV was recorded within the potential range of 0.2 to 0.6 V with a modulation amplitude of 0.05 V, a pulse width of 0.05 s, and sample width of 0.0167 s.
3. Results and discussion 3.1. Characterization of the prepared NP-PtTi alloy SEM, TEM, EDS, and XRD of the NP-PtTi alloy are shown in Fig. 1. SEM (Fig. 1A) shows that the dealloyed sample has interconnected spongy morphology with a ligament size less than 10 nm, which is useful for increasing the special surface areas (Duan et al., 2015). TEM image (Fig. 1B) provides more details on the structure. The clear contrast of the bright pores and the dark ligaments further confirms the formation of a 3D interconnected network structure, which is consistent with the SEM observation. The composition of the NP-PtTi alloy was monitored by EDS (Fig. 1C). The nanoporous material is composed of Pt with a small amount of Ti. XRD analysis (Fig. 1D) was used to examine the crystal structure of the dealloyed sample. Three diffraction peaks emerge around 40.1, 46.6, and 68.1, which could be assigned to reflections from the (111), (200), and (220) crystal planes for a face centered cubic (fcc) PtTi alloy, indicating that Ti atoms uniformly distributed into the fcc structure of Pt (Duan et al., 2015; Zhang
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Fig. 1. (A) SEM, (B) TEM, (C) EDS, and (D) XRD of NP-PtTi alloy.
et al., 2009; Hwang et al., 2015). Meanwhile, the grain size distribution and pore size distribution are shown in Fig. S1. The average size of grain was 77.6 nm and the pore size distribution is 5 nm. All the results indicate that NP-PtTi alloy with nanoporous structure has been fabricated successfully by one step dealloying procedure (Hwang et al., 2015; Liu et al., 2014). 3.2. SEM characterization of modified electrodes The morphologies of the different modified electrodes are shown in Fig. 2. The MWCNTs are highly entangled with one another according to previous report (Guo et al., 2014). In contrast, the surface morphology of MWCNTs–HMIMPF6/GCE reveals that the entangled MWCNTs bundles are stretched to show highly dispersed finer bundles (Fig. 2A), which can be ascribed to the strong π–π stacking interactions (Fukushima et al., 2003; Guo et al., 2014; Ma and Dougherty, 1997) and weak “cation–π” interaction (Fukushima et al., 2003; Guo et al., 2014; Bellayer et al., 2005) between MWCNTs and IL. [HMIM þ ] consists of imidazole ring and alkyl chain. Imidazole ring possesses π-conjugated structure, and the positive charge mainly localizes in imidazole ring. The π-electron and cation in HMIMPF6 interact with π-electron in the MWCNTs, which weakens the van der Waals interactions among the nanotubes. HMIMPF6 plays an important role in dispersing the MWCNTs (Guo et al., 2014). From Fig. 2B, a number
of bright dots, namely NP-PtTi alloy were obviously observed, suggesting the successful fabrication of NP-PtTi alloy on the surface of MWCNTs–HMIMPF6. When the kanamycin aptamer was assembled on the electrode, it shows that the surface morphology of aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE is different from that of NP-PtTi/MWCNTs–HMIMPF6/GCE (Fig. 2C), which confirms the attachment of the aptamer onto the modified electrode surface. Results mentioned above successfully demonstrated the mainly constructing process of the different modified electrodes. 3.3. Electrochemical characterization of the modified electrodes Different modified electrodes were characterized by CVs and EIS. Fig. 3 shows CVs of different modified electrodes in 5 mM Fe (CN)63 − /4 − containing 0.2 M KCl at a scan rate of 100 mV/s. A pair of well-defined reversible redox peaks was observed at the bare GCE. After MWCNTs–HMIMPF6 composite was immobilized on the electrode surface, the peak current of [Fe(CN)6]3 /4 redox couple was increased significantly due to the excellent electrical conductivity and larger special surface area of the composite. In order to investigate synergistic effect of the composite, CVs of NP-PtTi/GCE, and MWCNTs/GCE, MWCNTs–HMIMPF6/GCE, NP-PtTi/MWCNTs–HMIMPF6/GCE are shown in Fig. S2. The peak current of MWCNTs–HMIMPF6/GCE is larger than that of MWCNTs/GCE, which proves the improvement of dispersion of
Fig. 2. SEM images of (A) MWCNTs–HMIMPF6/GCE, (B) NP-PtTi/MWCNTs–HMIMPF6/GCE, and (C) aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE.
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Fig. 3. (A) CVs (scan rate: 100 mV/s) and (B) EIS of (a) bare GCE, (b) NP-PtTi/MWCNTs–HMIMPF6/GCE, (c) aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE, (d) BSA/aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE, and (e) kanamycin/BSA/aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE.
MWCNTs by HMIMPF6 (Fukushima et al., 2003; Guo et al., 2014). The peak current of the NP-PtTi/GCE shows a higher current in comparison with the bare GCE, suggesting that NP-PtTi alloy has excellent conductivity. Moreover, it can be clearly seen that the current of NP-PtTi/MWCNTs–HMIMPF6/GCE is superior to that of the MWCNTs MWCNTs–HMIMPF6/GCE and NP-PtTi/GCE. The composite of MWCNTs–HMIMPF6 and NP-PtTi alloy can increase the current signal significantly (Hou et al., 2013; Nie et al., 2012). MWCNTs–HMIMPF6 can bring about a sensitive electrode substrate and the introduction of NP-PtTi alloy can offer unprecedented benefits in catalysis design, which might be a novel idea for the construction of the electrochemical aptasensors. After the immobilization of the kanamycin aptamer on the electrode surface, an obvious decrease of redox peaks was obtained (curve c in Fig. 3A). Results indicate 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 was observed (curve d in Fig. 3A), suggesting the successful assembly of BSA as an isolating layer. Subsequently, the reaction between the aptamer and kanamycin led to a further decrease of current signal (curve e in Fig. 3A), indicating the formation of the aptamer–kanamycin complex hindered the interfacial electron transfer to the modified electrode surface. EIS is an effective method to further characterize the electron transfer properties of the different modified electrodes. In EIS, the semicircle portion at higher frequencies corresponds to the electron transfer process, and the linear portion at lower frequencies represents the diffusion process. The semicircle diameter of the Nyquist plot reflects the electron transfer resistance (Rct). Fig. 3B shows the Nyquist plots of various modified electrodes. It is observed that a relatively larger interface electron transfer resistance was obtained at the bare GCE. Compared with Rct of the bared electrode, Rct of the modified electrode was decreased. After the deposition of the MWCNTs–HMIMPF6 composite, Rct of the electrode decreased, indicating that MWCNTs–HMIMPF6 can accelerate the electron transfer. It is clear seen that Rct of NP-PtTi/MWCNTs–HMIMPF6/GCE was decreased compared with that of MWCNTs–HMIMPF6/GCE, implying that NP-PtTi/MWCNTs–HMIMPF6 nanocomposite can promote electron transfer and increase the electrode surface area. When aptamer was modified on the NP-PtTi/MWCNTs–HMIMPF6/GCE, Rct was increased, which can be attributed to the modification of the isolated biomolecules. Moreover, the capture of BSA and kanamycin resulted in the further increase of Rct, implying that electron transfer becomes more difficult. Results of EIS were agreed with those of the CVs. All those results prove that NP-PtTi/MWCNTs-HMIMPF6 nanocomposite not only offers a biocompatible surface for biomolecules loading, but also provides a sensitive electric interface for further sensing.
The electroactive surface areas (A) of the different kinds of electrodes were calculated based on the Randles–Sevcik equation (MansouriMajda et al., 2013; Elyasi et al., 2013): Ip¼ 2.65 105n3/2AD1/2υ1/2C, where Ip is the peak current, n is the transferring electron number, A is the electroactive area (cm2), D is the diffusion coefficient, υ is the scan rate, and C is the concentration of the substrate. The diffusion coefficient of K3[Fe(CN)6] is 7.6 10 6 cm2 s 1 (Bo et al., 2011). The calculated results are listed in Table S1. Results showed that values of A were calculated to be 0.0695 cm2, 0.187, 0.406 cm2, and 0.541 cm2 for GCE, MWCNTs/GCE, MWCNTs–HMIMPF6/GCE, and NP-PtTi/MWCNTs–HMIMPF6/GCE, respectively. A of MWCNTs–HMIMPF6 modified electrode is larger than that of MWCNTs modified electrode, which further proved that MWCNTs bundles can be considerably untangled within HMIMPF6. A of NP-PtTi alloy deposited MWCNTs–HMIMPF6/GCE is larger than that of the other modified electrodes, which proves the advantage of the composite of MWCNTs–HMIMPF6 and NP-PtTi alloy. 3.4. Optimization of experimental conditions In order to increase the sensitivity and selectivity of the aptasensor, experimental parameters such as the mass ratio of MWCNTs and HMIMPF6, incubation time, and pH value were studied systematically. As shown in Fig. S3A, when the ratio of MWCNTs and HMIMPF6 ranged from 1:6 to 1:8, the current signal increased insignificantly. The reason might be the solvent effect of IL. In the presence of HMIMPF6, MWCNTs are detached from the bundles and HMIMPF6 could keep the detached MWCNTs from rebinding together again by inhibiting the π–π stacking interaction between MWCNTs. Further increasing the amount of HMIMPF6, namely the ratio of MWCNTs and HMIMPF6 beyond 1:8, the current signal decreased on the contrary. If there was too much HMIMPF6, the conductive advantage of MWCNTs could be wrapped up by HMIMPF6. The highest value of electrochemical response was achieved at the ratio of 1:8. Therefore, HMIMPF6 plays an irreplaceable role in dispersing MWCNTs. The incubation time of kanamycin is an important parameter for response signal. A series of modified electrodes were incubated with 10 ng mL 1 kanamycin from 0 to 180 min. The difference between the DPV current intensity (ΔI) of aptamer/NP-PtTi/MWCNTs– HMIMPF6/GCE and that of kanamycin/BSA/aptamer/NP-PtTi/ MWCNTs–HMIMPF6/GCE was adopted to quantitatively detect kanamycin. In Fig. S3B, ΔI increases significantly with the increase in incubation time and tends to a steady value after 120 min, which suggests that the building of kanamycin–aptamer complex reaches the saturation. Therefore, 120 min was chosen as the optimized incubation time for the determination of kanamycin. The effect of the pH value of the measuring solution was also
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Fig. 4. Calibration curve of DPV peak currents for different kanamycin concentrations from 0.05 to 100 ng mL 1. The inset shows DPV responses of the electrochemical aptasensor to different concentrations of kanamycin (from a to j: 0, 0.05, 5, 10, 20, 30, 50, 70, 85, 100 ng mL 1).
investigated (Fig. S3C). ΔI increases with increasing pH value from 5.8 to 7.4, and then suddenly decreases at 7.4. Results suggest that strong acidic and alkaline solutions can damage the performance of aptamer and further affect the affinity between aptamer and the electrode surface. Thus, pH 7.4 was employed throughout the detection process. 3.5. Sensitivity of the aptasensor Under optimal conditions, the modified electrodes were incubated in different concentrations of kanamycin. As shown in Fig. 4, ΔI increases linearly with the increase in the concentration of kanamycin in the range of 0.05–100 ng mL 1 with a correlation coefficient of 0.9991. The regression equation was ΔI (μA)¼ 4.31049 c þ8.84992 (ng mL 1). A detection limit of 3.7 pg mL 1 was achieved (S/N ¼3). The low limit of detection can be considered as followed: (1) HMIMPF6 acts not only as a good solvent but also as a suitable charge-transfer bridge to facilitate the electrode transfer rate. The synergy of the MWCNTs and IL can greatly enhance the conductivity; (2) MWCNTs–HMIMPF6/NP-PtTi alloy composite acts as an effective load matrix for electronic transfer; (3) NP-PtTi alloy with high surface-to-volume ratio can enhance the immobilized amount of kanamycin aptamer and significantly improve the sensitivity of kanamycin detection. From Table S2, we can see that compared with other methods, the as-proposed method has a relatively high sensitivity and low detection limit. 3.6. The stability, specificity and reproducibility of the aptasensor To evaluate the stability of the aptasensor, five electrodes were independently prepared and stored at 4 °C before use. After two weeks, the response of the aptasensor for the detection of kanamycin was retained at 94% of the initial response. The aptasensor kept long-term stability, which was ascribed to the stability of MWNCTs-HMIMPF6/NP-PtTi. Specificity is another important criterion for electrochemical aptasensors. The current responses of the as-prepared aptasensors to 10 ng mL 1 solutions of kanamycin, glucose, ascorbic acid, uric acid, methionine, and the mixture of 10 ng mL 1 kanamycin solution and 20 ng mL 1 interfering substance solutions were studied. As shown in Fig. 5, the aptasensor in kanamycin solution shows a much stronger current response (Fig. 5a) compared with
Fig. 5. DPV current responses of the aptasensor to (a) kanamycin, (b–e) interferents, and (f–i) mixtures of kanamycin and different interferents. Error bars are standard deviations across three repetitive experiments.
that produced in other interfering substance solutions. Then, it is noticeable that a weak current response was obtained in the presence of those interferents (Fig. 5b–e), proving the special identification of kanamycin aptamer. In addition, mixtures of kanamycin and interferents also showed a much stronger current response (Fig. 5f–i). All those results confirmed that the as-prepared aptasensor had high selectivity. To test the reproducibility of the aptasensor, five electrodes were prepared in the same way to measure 10 ng mL 1 kanamycin solution. The relative standard deviation (RSD) of the measurements was 3.1%, indicating the reproducibility of the aptasensor is acceptable. 3.7. Determination of kanamycin in real samples Although the prepared aptasensor displayed excellent selectivity towards kanamycin, it is worthy to demonstrate the possibility of the proposed aptasensor for practical application. Mike is one of the most important regulated products in food analysis due to the risk of having veterinary medicine residue. Thus, it has attracted considerable attention to detect kanamycin in milk. Kanamycin standard solution was spiked into the 10 diluted milk with PBS to prepare the kanamycin concentrations of 0.5, 0.8, 1.0, 5.0, and 8.0 ng mL 1. Five independent aptasensors were used to detect the different concentrations of kanamycin. Results are shown in Table S3. The recovery of kanamycin concentration ranges from 94.66% to 108.95%. To further investigate the practical application of the aptasensor, a controlled trial by a reference ELISA (W. Xu et al., 2014) method was performed. The relative deviation between the two methods ranges from 1.84% to 4.8%, indicating that there is no significant difference between the electrochemical and ELISA methods. Therefore, the method used in this paper can be successfully applied to detect kanamycin in milk sample.
4. Conclusion In this work, an ultrasensitive aptasensor based on NP-PtTi/ MWNCTs-HMIMPF6 as a sensor platform to sequentially immobilize kanamycin aptamer was designed for kanamycin detection. The prepared aptasensor offered several advantages: (1) The MWCNTs–HMIMPF6/NP-PtTi nanocomposite not only improved the conductivity of electrodes, but also enhanced the immobilized
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quantity of biomolecules; (2) The constructed aptasensor with higher sensitivity exhibits a wide linear range for kanamycin from 0.05 to 100 ng mL 1 with a low detection limit of 3.7 pg mL 1; (3) The constructed aptasensor offers the advantages of improved stability, specificity and reproducibility. In addition, the resulting aptasensor was successfully applied for kanamycin detection in real milk samples. Thus, the presented aptasensor provides the potential applications for kanamycin detection in the field of food analysis.
Acknowledgments 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 material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.06.081.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel electrochemical aptasensor for ultrasensitive detection of kanamycin based on MWCNTs–HMIMPF6 and nanoporous PtTi alloy Wenjuan Guo a, Na Sun b, Xiaoli Qin a, Meishan Pei a,n, Luyan Wang a a Shandong Provincial Key Laboratory of Chemical Sensing & Analysis, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China b Environmental Protection Monitoring Station, Jining 272045, China
art ic l e i nf o Article history: Received 16 April 2015 Received in revised form 22 June 2015 Accepted 23 June 2015 Available online 10 July 2015 Keywords: Aptasensor Ionic liquid Nanoporous Platinum Kanamycin
a b s t r a c t A novel aptasensor based on a novel composite film consisting of multi-walled carbon nanotubes (MWCNTs), ionic liquid (IL) of 1-hexyl-3-methylimidazolium hexafluorophosphate (HMIMPF6), and nanoporous PtTi (NPPtTi) alloy was constructed for ultrasensitive detection of kanamycin. The NP-PtTi alloy was successfully fabricated by a simple dealloying of PtTiAl source alloy in HCl solution. The NP-PtTi alloy has uniform interconnected network structure with specific surface area and was used to immobilize aptamer. After modified with the composite material, current signal was amplified obviously, which attributed to the larger specific surface area and excellent electrical conductivity of NP-PtTi and MWCNTs. A number of factors affecting the activity of the aptasensor have been discussed and optimized. Under the optimized conditions, the proposed aptasensor provided a linear range of 0.05–100 ng mL 1 with a low detection limit of 3.7 pg mL 1. This aptasensor displayed high sensitivity, stability and reproducibility. In addition, the as-prepared aptasensor was successfully used for the determination of kanamycin in a real sample. & 2015 Published by Elsevier B.V.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Reagents and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fabrication of NP-PtTi alloy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Construction of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Electrochemical measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Characterization of the prepared NP-PtTi alloy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SEM characterization of modified electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Electrochemical characterization of the modified electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Optimization of experimental conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Sensitivity of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. The stability, specificity and reproducibility of the aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Determination of kanamycin in real samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author. Fax: þ 86 531 82765475. E-mail address: [email protected] (M. Pei).
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1. Introduction Kanamycin is an aminoglycoside antibiotic which is produced by the fermentation of streptomyces kanamyceticus (Spahn and Prescott, 1996; Bai et al., 2014). It is used to treat a variety of infections by inducing mistranslation and indirectly inhibiting translocation during protein synthesis (Wirmer and Westhof 2006; Song et al., 2011). The abuse of kanamycin can cause serious side effects, such as loss of hearing, toxicity to the kidneys, and allergic reactions to the drugs (Bai et al., 2014; Wirmer and Westhof 2006). To protect the customers’ security, the European Union (EU) has established maximum residue limits (MRLs) for kanamycin in food, such as 100 μg kg 1 for meat, 600 μg kg 1 for liver, 2500 μg kg 1 for kidney, and 150 μg kg 1 for milk ( Scheme 1). At present, many analytical methods have been reported for the detection of kanamycin. A label-free immunosensor was constructed based on nanoparticles and graphene sheet mixed with thionine (Yu et al., 2013). An immunosensor was fabricated based on water-soluble graphene sheet/prussian blue-chitosan/nanoporous gold composited film modified electrode (Zhao et al., 2011). Chen et al. (2013) prepared an immunosensor based on MRS assay and bovine serum albumin (BSA) system. An electrochemical aptasensor based on the synergistic contributions of multiple nanocomposites for kanamycin detection has been reported (Sun et al., 2014). The constructing process of the sensors motioned above usually involved complicated assembly processes, and the usage of a large amount of reagents. The quick determination of kanamycin with low cost is still a challenge in the practical applications. It is essential to develop the applicable techniques for simple, low-cost and sensitive detections. Aptamers, the synthetic single-stranded DNA or RNA molecules with specific three-dimensional (3D) structures, are selected in vitro through systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, 1990; Zhu et al., 2012). Aptamers can be used as the recognition probes superior to antibodies because of their advantages such as good stability, easy modification, high affinity and specificity (Wei et al., 2012). Thus, the electrochemical aptasensors are especially attractive in the fields of food safety and clinical diagnosis. Recently, aptasensors (Zhu et al., 2012; W. Xu et al., 2014) have been used in many field, such as chemiluminescence (Yan et al., 2012; Yu et al., 2011a, 2011b), electrochemistry (Sun et al., 2014), colorimetric (Wang et al., 2012), and immunoassay (Ge et al., 2012a, 2012b; Zang et al.,
2012). Among them, the electrochemical detection based on aptasensor has shown significant advantages, such as high sensitivity, fast response time, simple operation, and low cost. The signal amplification is a key factor for the construction of aptasensor. Strategies based on nanomaterials have promising potential in ultrasensitive biological detection due to the versatile properties of nanomaterials. Various nanomaterials including Au, Ag, Cu, Pd, and Pt-based metals have been used in constructing biosensors with high sensitivity (Wang et al., 2014; Liu et al., 2013; Chen et al., 2012). At present, the nanoporous materials are particularly attractive in bioassays due to their large specific surface area. Nanoporous Pt-based alloy exhibited more active sites than the single Pt, Pd, and Au nanoparticles (Crumbliss et al., 1992; Xu et al., 2010). PtTi alloy nanoparticles of various ratios were prepared by using Pt2(dba)3 and titanium chloride (TiCl4) as precursors (Ding et al., 2008). Results showed that PtTi/C alloy nanoparticles showed higher activity than Pt/C catalyst. PtTi-based ternary alloy was found to exhibit higher activity than pure Pt electrocatalyst (He and Kreidler, 2008). A nanoporous PtTi alloy with multimodal ligament/pore structure was designed, which showed high activity and high catalytic durability (Duan et al., 2015). Based on that point, in this paper, nanoporous Pt–Ti (NPPtTi) alloy with uniform nanoporous structure was successfully fabricated by selectively dealloying Al from a PtTiAl alloy in HCl solution. NP-PtTi alloy with excellent electrical conductivity has three-dimensional (3D) interconnected network structure, which increases its large specific surface area and provides a beneficial immobilization platform for kanamycin aptamer carrier. Till now, carbon nanotubes (CNTs) have gained increasing interests in a great variety of research fields (Gopalan et al., 2009; Tunckol et al., 2013; Liu et al., 2011) due to their high electrical conductivity, chemical stability, and remarkable mechanical strength. Even though MWCNTs were hydrophobic and difficult to be dispersed by ordinary solvents, many approaches have been developed to overcome the above obstacle, including sidewall functionalization (Holzinger et al., 2003), polymer wrapping (Zheng et al., 2003), and addition of surfactants (Moore et al., 2003). However, these methods did not avoid some drawbacks, such as time-consuming, complicated physical and chemical reaction. With the development of IL, it is found that CNTs could be easily untangled into much finer bundles in the IL (Xiao et al., 2008). Aida’s group (Fukushima et al., 2003) prepared a gel containing pristine single-walled carbon nanotubes mixed with IL to
Scheme 1. Schematic diagram of the kanamycin aptasensor.
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improve the performance of electrodes. Our group (Guo et al., 2014) also found the synergic effect of the MWCNTs and IL. Results showed that MWCNTs–IL composite could be used as an effective load matrix and the enhancement effect of the current response is significant, which provided a new way for constructing the powerful biosensors with excellent properties. In this paper, a novel electrochemical sensor for ultrasensitive detection of kanamycin was fabricated based on MWCNTs–1-hexyl3-methylimidazolium hexafluorophosphate (HMIMPF6) (MWCNTs– HMIMPF6) and NP-PtTi alloy as the matrix. MWCNTs–HMIMPF6/NPPtTi alloy nanocomposite constructed an effective biomolecules immobilization matrix with unblocked conductive pathway for electron transfer. More importantly, MWCNTs–HMIMPF6/NP-PtTi nanocomposite was first used in electrochemical sensors as well with significant signal improvement. Under the optimum conditions, the prepared aptasensor had a wide linear response range and a lower detection limit, which proved this method to be an efficient method of constructing electrochemical aptasensor with highly sensitivity and specificity for the quantitative detection of kanamycin. In addition, the results from the electrochemical measurement were compared with that from ELISA method (W. Xu et al., 2014; Chen et al., 2013) for determining kanamycin in milk, which proved the as-prepared aptasensor was a useful tool for the detection of kanamycin in real samples.
2. Materials and methods 2.1. Reagents and materials A kanamycin aptamer modified with an amino group at the 5′ position, namely 5′-NH2-AGATGGGGGTTGAGGCTAAGCCGA-3′ was synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Kanamycin aptamer was selected by systematic evolution of ligands by exponential enrichment (SELEX) that could specifically bind with kanamycin (Zhu et al., 2012; W. Xu et al., 2014). MWCNTs were obtained from Beijing Dekedaojin technology Co., Ltd. (China). HMIMPF6, chitosan (CS), bovine serum albumin (BSA), glucose, ascorbic acid, uric acid, methionine, and kanamycin 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). Ultrapure water was used throughout the experiment. The concentration of kanamycin obtained based on the enzyme-linked immunosorbent assay (ELISA) method was provided by the Aladdin Chemical Reagent Co., Ltd. and the previous papers (Zhu et al., 2012; Sun et al., 2014). ELISA is a popular format of analytic biochemistry assay that uses an antibody to detect the presence of an antigen. The basic principle of an ELISA is to detect signal produced by enzyme which is linked to the antibody. 2.2. Apparatus Electrochemical experiments of CV and DPV were carried out with a CHI 760E electrochemical workstation (Chenhua Instruments Co., Shanghai, China). EIS was performed with a Zennium electrochemical workstation (Zahner, Germany). The morphologies and energy-dispersive X-ray spectroscopy (EDS) of the samples were characterized by a QUANTA PEG 250 field emission scanning electron microscope (SEM). Transmission electron microscopy (TEM) was obtained with a JEM-2100 high resolution transmission electron microscope. Powder X-ray diffraction (XRD) data were obtained on a Bruker D8 advanced X-ray diffractometer using Cu Kα radiation at a scan of 0.04°/s. Grain size distribution of NP-PtTi alloy was tested by a laser particle size analyzer (Britain, Malvern).
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2.3. Fabrication of NP-PtTi alloy Pt10Ti10Al80 ternary alloy foils were prepared by refining the corresponding metals Pt, Ti, and Al (99.99%) under an Ar-protected atmosphere in an arc-furnace, followed by melt-spinning (Duan et al., 2015; C.X. Xu et al., 2014; Zhang et al., 2009). Pt10Ti10Al80 alloy foils were dealloyed in 1 M HCl solution at room temperature (RT) for 30 min to obtain NP-PtTi alloy by etching the Al atoms and part of Ti atoms. After dealloying, the product was washed several times with ultrapure water and dried at RT in air. 2 mg NP-PtTi alloys were dispersed in 1 mL of 1 wt% CS under sonication to obtain a homogeneous suspension. 2.4. Construction of the aptasensor The bared GCE was polished with 0.3 and 0.05 μm alumina slurry, and then thoroughly washed in ethanol and ultrapure water, respectively. MWCNTs–HMIMPF6 composite was fabricated according to the literature (Guo et al., 2014). 5 μL MWCNTs–HMIMPF6 suspensions were dropped onto the surface of the GCE, and then dried directly in air. Next, 5 μL NP-PtTi suspensions were dropped onto the MWCNTs–HMIMPF6 modified electrode surface. After washing with the PBS (pH ¼ 7.4), the modified electrode was immersed into 10 μL of 5 μmol L 1 aptamer solution overnight. Then the electrode was washed with PBS to remove the unbound aptamer. 10 μL of 1% BSA solution were employed to block nonspecific binding sites for 2 h at 4 °C. After washed by PBS thoroughly several times, the modified electrode was incubated in 10 mL kanamycin solution with different concentrations for 2 h. The as-prepared electrode was stored in the refrigerator prior to use. The schematic diagram of the kanamycin aptasensor is shown in Fig. 1. 2.5. Electrochemical measurements All electrochemical measurements were performed using a conventional three-electrode system composed of a GCE as the working electrode, a platinum wire as the counter electrode, and a KCl saturated Ag/AgCl as the reference electrode. The EIS measurement was performed in the presence of 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) and 0.2 M KCl mixture in PBS (pH¼7.4). DPV was recorded within the potential range of 0.2 to 0.6 V with a modulation amplitude of 0.05 V, a pulse width of 0.05 s, and sample width of 0.0167 s.
3. Results and discussion 3.1. Characterization of the prepared NP-PtTi alloy SEM, TEM, EDS, and XRD of the NP-PtTi alloy are shown in Fig. 1. SEM (Fig. 1A) shows that the dealloyed sample has interconnected spongy morphology with a ligament size less than 10 nm, which is useful for increasing the special surface areas (Duan et al., 2015). TEM image (Fig. 1B) provides more details on the structure. The clear contrast of the bright pores and the dark ligaments further confirms the formation of a 3D interconnected network structure, which is consistent with the SEM observation. The composition of the NP-PtTi alloy was monitored by EDS (Fig. 1C). The nanoporous material is composed of Pt with a small amount of Ti. XRD analysis (Fig. 1D) was used to examine the crystal structure of the dealloyed sample. Three diffraction peaks emerge around 40.1, 46.6, and 68.1, which could be assigned to reflections from the (111), (200), and (220) crystal planes for a face centered cubic (fcc) PtTi alloy, indicating that Ti atoms uniformly distributed into the fcc structure of Pt (Duan et al., 2015; Zhang
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Fig. 1. (A) SEM, (B) TEM, (C) EDS, and (D) XRD of NP-PtTi alloy.
et al., 2009; Hwang et al., 2015). Meanwhile, the grain size distribution and pore size distribution are shown in Fig. S1. The average size of grain was 77.6 nm and the pore size distribution is 5 nm. All the results indicate that NP-PtTi alloy with nanoporous structure has been fabricated successfully by one step dealloying procedure (Hwang et al., 2015; Liu et al., 2014). 3.2. SEM characterization of modified electrodes The morphologies of the different modified electrodes are shown in Fig. 2. The MWCNTs are highly entangled with one another according to previous report (Guo et al., 2014). In contrast, the surface morphology of MWCNTs–HMIMPF6/GCE reveals that the entangled MWCNTs bundles are stretched to show highly dispersed finer bundles (Fig. 2A), which can be ascribed to the strong π–π stacking interactions (Fukushima et al., 2003; Guo et al., 2014; Ma and Dougherty, 1997) and weak “cation–π” interaction (Fukushima et al., 2003; Guo et al., 2014; Bellayer et al., 2005) between MWCNTs and IL. [HMIM þ ] consists of imidazole ring and alkyl chain. Imidazole ring possesses π-conjugated structure, and the positive charge mainly localizes in imidazole ring. The π-electron and cation in HMIMPF6 interact with π-electron in the MWCNTs, which weakens the van der Waals interactions among the nanotubes. HMIMPF6 plays an important role in dispersing the MWCNTs (Guo et al., 2014). From Fig. 2B, a number
of bright dots, namely NP-PtTi alloy were obviously observed, suggesting the successful fabrication of NP-PtTi alloy on the surface of MWCNTs–HMIMPF6. When the kanamycin aptamer was assembled on the electrode, it shows that the surface morphology of aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE is different from that of NP-PtTi/MWCNTs–HMIMPF6/GCE (Fig. 2C), which confirms the attachment of the aptamer onto the modified electrode surface. Results mentioned above successfully demonstrated the mainly constructing process of the different modified electrodes. 3.3. Electrochemical characterization of the modified electrodes Different modified electrodes were characterized by CVs and EIS. Fig. 3 shows CVs of different modified electrodes in 5 mM Fe (CN)63 − /4 − containing 0.2 M KCl at a scan rate of 100 mV/s. A pair of well-defined reversible redox peaks was observed at the bare GCE. After MWCNTs–HMIMPF6 composite was immobilized on the electrode surface, the peak current of [Fe(CN)6]3 /4 redox couple was increased significantly due to the excellent electrical conductivity and larger special surface area of the composite. In order to investigate synergistic effect of the composite, CVs of NP-PtTi/GCE, and MWCNTs/GCE, MWCNTs–HMIMPF6/GCE, NP-PtTi/MWCNTs–HMIMPF6/GCE are shown in Fig. S2. The peak current of MWCNTs–HMIMPF6/GCE is larger than that of MWCNTs/GCE, which proves the improvement of dispersion of
Fig. 2. SEM images of (A) MWCNTs–HMIMPF6/GCE, (B) NP-PtTi/MWCNTs–HMIMPF6/GCE, and (C) aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE.
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Fig. 3. (A) CVs (scan rate: 100 mV/s) and (B) EIS of (a) bare GCE, (b) NP-PtTi/MWCNTs–HMIMPF6/GCE, (c) aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE, (d) BSA/aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE, and (e) kanamycin/BSA/aptamer/NP-PtTi/MWCNTs–HMIMPF6/GCE.
MWCNTs by HMIMPF6 (Fukushima et al., 2003; Guo et al., 2014). The peak current of the NP-PtTi/GCE shows a higher current in comparison with the bare GCE, suggesting that NP-PtTi alloy has excellent conductivity. Moreover, it can be clearly seen that the current of NP-PtTi/MWCNTs–HMIMPF6/GCE is superior to that of the MWCNTs MWCNTs–HMIMPF6/GCE and NP-PtTi/GCE. The composite of MWCNTs–HMIMPF6 and NP-PtTi alloy can increase the current signal significantly (Hou et al., 2013; Nie et al., 2012). MWCNTs–HMIMPF6 can bring about a sensitive electrode substrate and the introduction of NP-PtTi alloy can offer unprecedented benefits in catalysis design, which might be a novel idea for the construction of the electrochemical aptasensors. After the immobilization of the kanamycin aptamer on the electrode surface, an obvious decrease of redox peaks was obtained (curve c in Fig. 3A). Results indicate 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 was observed (curve d in Fig. 3A), suggesting the successful assembly of BSA as an isolating layer. Subsequently, the reaction between the aptamer and kanamycin led to a further decrease of current signal (curve e in Fig. 3A), indicating the formation of the aptamer–kanamycin complex hindered the interfacial electron transfer to the modified electrode surface. EIS is an effective method to further characterize the electron transfer properties of the different modified electrodes. In EIS, the semicircle portion at higher frequencies corresponds to the electron transfer process, and the linear portion at lower frequencies represents the diffusion process. The semicircle diameter of the Nyquist plot reflects the electron transfer resistance (Rct). Fig. 3B shows the Nyquist plots of various modified electrodes. It is observed that a relatively larger interface electron transfer resistance was obtained at the bare GCE. Compared with Rct of the bared electrode, Rct of the modified electrode was decreased. After the deposition of the MWCNTs–HMIMPF6 composite, Rct of the electrode decreased, indicating that MWCNTs–HMIMPF6 can accelerate the electron transfer. It is clear seen that Rct of NP-PtTi/MWCNTs–HMIMPF6/GCE was decreased compared with that of MWCNTs–HMIMPF6/GCE, implying that NP-PtTi/MWCNTs–HMIMPF6 nanocomposite can promote electron transfer and increase the electrode surface area. When aptamer was modified on the NP-PtTi/MWCNTs–HMIMPF6/GCE, Rct was increased, which can be attributed to the modification of the isolated biomolecules. Moreover, the capture of BSA and kanamycin resulted in the further increase of Rct, implying that electron transfer becomes more difficult. Results of EIS were agreed with those of the CVs. All those results prove that NP-PtTi/MWCNTs-HMIMPF6 nanocomposite not only offers a biocompatible surface for biomolecules loading, but also provides a sensitive electric interface for further sensing.
The electroactive surface areas (A) of the different kinds of electrodes were calculated based on the Randles–Sevcik equation (MansouriMajda et al., 2013; Elyasi et al., 2013): Ip¼ 2.65 105n3/2AD1/2υ1/2C, where Ip is the peak current, n is the transferring electron number, A is the electroactive area (cm2), D is the diffusion coefficient, υ is the scan rate, and C is the concentration of the substrate. The diffusion coefficient of K3[Fe(CN)6] is 7.6 10 6 cm2 s 1 (Bo et al., 2011). The calculated results are listed in Table S1. Results showed that values of A were calculated to be 0.0695 cm2, 0.187, 0.406 cm2, and 0.541 cm2 for GCE, MWCNTs/GCE, MWCNTs–HMIMPF6/GCE, and NP-PtTi/MWCNTs–HMIMPF6/GCE, respectively. A of MWCNTs–HMIMPF6 modified electrode is larger than that of MWCNTs modified electrode, which further proved that MWCNTs bundles can be considerably untangled within HMIMPF6. A of NP-PtTi alloy deposited MWCNTs–HMIMPF6/GCE is larger than that of the other modified electrodes, which proves the advantage of the composite of MWCNTs–HMIMPF6 and NP-PtTi alloy. 3.4. Optimization of experimental conditions In order to increase the sensitivity and selectivity of the aptasensor, experimental parameters such as the mass ratio of MWCNTs and HMIMPF6, incubation time, and pH value were studied systematically. As shown in Fig. S3A, when the ratio of MWCNTs and HMIMPF6 ranged from 1:6 to 1:8, the current signal increased insignificantly. The reason might be the solvent effect of IL. In the presence of HMIMPF6, MWCNTs are detached from the bundles and HMIMPF6 could keep the detached MWCNTs from rebinding together again by inhibiting the π–π stacking interaction between MWCNTs. Further increasing the amount of HMIMPF6, namely the ratio of MWCNTs and HMIMPF6 beyond 1:8, the current signal decreased on the contrary. If there was too much HMIMPF6, the conductive advantage of MWCNTs could be wrapped up by HMIMPF6. The highest value of electrochemical response was achieved at the ratio of 1:8. Therefore, HMIMPF6 plays an irreplaceable role in dispersing MWCNTs. The incubation time of kanamycin is an important parameter for response signal. A series of modified electrodes were incubated with 10 ng mL 1 kanamycin from 0 to 180 min. The difference between the DPV current intensity (ΔI) of aptamer/NP-PtTi/MWCNTs– HMIMPF6/GCE and that of kanamycin/BSA/aptamer/NP-PtTi/ MWCNTs–HMIMPF6/GCE was adopted to quantitatively detect kanamycin. In Fig. S3B, ΔI increases significantly with the increase in incubation time and tends to a steady value after 120 min, which suggests that the building of kanamycin–aptamer complex reaches the saturation. Therefore, 120 min was chosen as the optimized incubation time for the determination of kanamycin. The effect of the pH value of the measuring solution was also
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Fig. 4. Calibration curve of DPV peak currents for different kanamycin concentrations from 0.05 to 100 ng mL 1. The inset shows DPV responses of the electrochemical aptasensor to different concentrations of kanamycin (from a to j: 0, 0.05, 5, 10, 20, 30, 50, 70, 85, 100 ng mL 1).
investigated (Fig. S3C). ΔI increases with increasing pH value from 5.8 to 7.4, and then suddenly decreases at 7.4. Results suggest that strong acidic and alkaline solutions can damage the performance of aptamer and further affect the affinity between aptamer and the electrode surface. Thus, pH 7.4 was employed throughout the detection process. 3.5. Sensitivity of the aptasensor Under optimal conditions, the modified electrodes were incubated in different concentrations of kanamycin. As shown in Fig. 4, ΔI increases linearly with the increase in the concentration of kanamycin in the range of 0.05–100 ng mL 1 with a correlation coefficient of 0.9991. The regression equation was ΔI (μA)¼ 4.31049 c þ8.84992 (ng mL 1). A detection limit of 3.7 pg mL 1 was achieved (S/N ¼3). The low limit of detection can be considered as followed: (1) HMIMPF6 acts not only as a good solvent but also as a suitable charge-transfer bridge to facilitate the electrode transfer rate. The synergy of the MWCNTs and IL can greatly enhance the conductivity; (2) MWCNTs–HMIMPF6/NP-PtTi alloy composite acts as an effective load matrix for electronic transfer; (3) NP-PtTi alloy with high surface-to-volume ratio can enhance the immobilized amount of kanamycin aptamer and significantly improve the sensitivity of kanamycin detection. From Table S2, we can see that compared with other methods, the as-proposed method has a relatively high sensitivity and low detection limit. 3.6. The stability, specificity and reproducibility of the aptasensor To evaluate the stability of the aptasensor, five electrodes were independently prepared and stored at 4 °C before use. After two weeks, the response of the aptasensor for the detection of kanamycin was retained at 94% of the initial response. The aptasensor kept long-term stability, which was ascribed to the stability of MWNCTs-HMIMPF6/NP-PtTi. Specificity is another important criterion for electrochemical aptasensors. The current responses of the as-prepared aptasensors to 10 ng mL 1 solutions of kanamycin, glucose, ascorbic acid, uric acid, methionine, and the mixture of 10 ng mL 1 kanamycin solution and 20 ng mL 1 interfering substance solutions were studied. As shown in Fig. 5, the aptasensor in kanamycin solution shows a much stronger current response (Fig. 5a) compared with
Fig. 5. DPV current responses of the aptasensor to (a) kanamycin, (b–e) interferents, and (f–i) mixtures of kanamycin and different interferents. Error bars are standard deviations across three repetitive experiments.
that produced in other interfering substance solutions. Then, it is noticeable that a weak current response was obtained in the presence of those interferents (Fig. 5b–e), proving the special identification of kanamycin aptamer. In addition, mixtures of kanamycin and interferents also showed a much stronger current response (Fig. 5f–i). All those results confirmed that the as-prepared aptasensor had high selectivity. To test the reproducibility of the aptasensor, five electrodes were prepared in the same way to measure 10 ng mL 1 kanamycin solution. The relative standard deviation (RSD) of the measurements was 3.1%, indicating the reproducibility of the aptasensor is acceptable. 3.7. Determination of kanamycin in real samples Although the prepared aptasensor displayed excellent selectivity towards kanamycin, it is worthy to demonstrate the possibility of the proposed aptasensor for practical application. Mike is one of the most important regulated products in food analysis due to the risk of having veterinary medicine residue. Thus, it has attracted considerable attention to detect kanamycin in milk. Kanamycin standard solution was spiked into the 10 diluted milk with PBS to prepare the kanamycin concentrations of 0.5, 0.8, 1.0, 5.0, and 8.0 ng mL 1. Five independent aptasensors were used to detect the different concentrations of kanamycin. Results are shown in Table S3. The recovery of kanamycin concentration ranges from 94.66% to 108.95%. To further investigate the practical application of the aptasensor, a controlled trial by a reference ELISA (W. Xu et al., 2014) method was performed. The relative deviation between the two methods ranges from 1.84% to 4.8%, indicating that there is no significant difference between the electrochemical and ELISA methods. Therefore, the method used in this paper can be successfully applied to detect kanamycin in milk sample.
4. Conclusion In this work, an ultrasensitive aptasensor based on NP-PtTi/ MWNCTs-HMIMPF6 as a sensor platform to sequentially immobilize kanamycin aptamer was designed for kanamycin detection. The prepared aptasensor offered several advantages: (1) The MWCNTs–HMIMPF6/NP-PtTi nanocomposite not only improved the conductivity of electrodes, but also enhanced the immobilized
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quantity of biomolecules; (2) The constructed aptasensor with higher sensitivity exhibits a wide linear range for kanamycin from 0.05 to 100 ng mL 1 with a low detection limit of 3.7 pg mL 1; (3) The constructed aptasensor offers the advantages of improved stability, specificity and reproducibility. In addition, the resulting aptasensor was successfully applied for kanamycin detection in real milk samples. Thus, the presented aptasensor provides the potential applications for kanamycin detection in the field of food analysis.
Acknowledgments 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 material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.06.081.
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