Analytica Chimica Acta 817 (2014) 33–41
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles Adem Zengin a,b , Ugur Tamer b , Tuncer Caykara a,∗ a b
Gazi University, Faculty of Science, Department of Chemistry, Besevler, 06500 Ankara, Turkey Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, Etiler, 06330 Ankara, Turkey
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Kanamycin immobilized hybrid magnetic nanoparticles were fabricated.
• They were sandwiched with the SERS substrate.
• The limit of detection was determined to be 2 pg mL−1 .
a r t i c l e
i n f o
Article history: Received 12 October 2013 Received in revised form 9 January 2014 Accepted 19 January 2014 Available online 30 January 2014 Keywords: Superparamagnetic nanoparticles Polymer brushes Kanamycin Surface enhanced Raman spectroscopy
a b s t r a c t Herein, we report the development of extremely sensitive sandwich assay of kanamycin using a combination of anti-kanamycin functionalized hybrid magnetic (Fe3 O4 ) nanoparticles (MNPs) and 2-mercaptobenzothiazole labeled Au-core@Ag-shell nanoparticles as the recognition and surfaceenhanced Raman scattering (SERS) substrate, respectively. The hybrid MNPs were first prepared via surface-mediated RAFT polymerization of N-acryloyl-l-glutamic acid in the presence of 2(butylsulfanylcarbonylthiolsulfanyl) propionic acid-modified MNPs as a RAFT agent and then biofunctionalized with anti-kanamycin, which are both specific for kanamycin and can be collected via a simple magnet. After separating kanamycin from the sample matrix, they were sandwiched with the SERS substrate. According to our experimental results, the limit of detection (LOD) was determined to be 2 pg mL−1 , this value being about 3–7 times more than sensitive than the LOD of previously reported results, which can be explained by the higher SERS activity of silver coated gold nanoparticles. The analysis time took less than 10 min, including washing and optical detection steps. Furthermore, the sandwich assay was evaluated for investigating the kanamycin specificity on neomycin, gentamycin and streptomycin and detecting kanamycin in artificially contaminated milk. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Kanamycin is an aminoglycoside antibiotic, which is fabricated by the fermentation of Streptomyces kanamyceticus and utilized to treat wide variety of infections by inducing mistranslation
∗ Corresponding author. Tel.: +90 312 202 11 24; fax: +90 312 212 22 79. E-mail address: [email protected] (T. Caykara). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.042
and indirectly inhibiting translocation during protein synthesis [1]. Similar to other aminoglycosides, kanamycin shows comparatively narrow safety margin and may cause many side effects such as loss of hearing, toxicity to kidneys, and allergic reactions to drugs [2] Moreover, the residual amount of kanamycin in the foodstuff may lead to antibiotic resistance from the pathogenic bacterial strains, which can endanger the consumer [3]. Hence, it is critical to developed sufficiently sensitive methods to detect kanamycin residue for clinical diagnosis and food safety. Recently,
34
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
a number of analytical methods, such as high performance liquid chromatography [4,5], label-free electrochemical immunosensor [6,7], capillary electrophoresis [8], enzyme-linked immunosorbent assay [9–12], surface plasmon resonance [13,14] and the microbiological multi-residue system [15] have been reported for the detection of kanamycin. However, most of those above-mentioned methods are often time consuming, tedious, and require great amount of reagents, solvents, and expensive apparatus. Thus, it is desirable to develop a low cost, selective and sensitive method for kanamycin detection with less reagent consumption. Recently, SERS-based assay method using antibody conjugated metal nanoparticles has attracted great interest from many researchers because of its rapid end sensitive sensing capability. >Moreover, most of them have focused on the application of Au nanoparticles SERS probes to assay systems [16,17]. Although Au nanoparticles are stable, biocompatible, they only exhibit a moderate enhancement in SERS detection compared to Ag nanoparticles [18]. However, the application of those Ag nanoparticles is more or less limited on account of their easily aggregations in solutions [19]. Therefore, bimetallic Au@Ag or Ag@Au core–shell nanoparticles have become the focus of attention due to their greater SERS activity in comparison with individual Ag and Au nanoparticles [20–25]. In practical terms, the Ag@Au structure has shown many drawbacks, high suppression of the optical properties of Ag by the Au shell, as well as significant challenges in preparing a probe with uniform structure [24,25]. In many cases, the relatively high reduction potential of Au causes Ag to be oxidized in the coating process, leading to structures with hollow cores or formation of non-continuous Au shell [25–27]. They are also inherently unstable due to the exposure of Ag to the outside environment, making them non-ideal for biomolecular sensing and diagnostics. However, the Au@Ag nanoparticle system is still very attractive as a platform for sensing applications. In this report, we present an extremely sensitive detection method for kanamycin utilizing anti-kanamycin functionalized hybrid MNPs and 2-mercaptobenzothiazole (MBT) labeled Au@Ag core–shell nanoparticles as probe and SERS substrate, respectively. Furthermore, this method was also tested for the kanamycin specificity on neomycin, gentamycin and streptomycin, and for the detection of kanamycin in artificially contaminated milk. The results undoubtedly demonstrated the superiority of SERS-based sandwich assay with extremely increased sensitivity using bimetallic Au@Ag core–shell nanoparticles.
2. Experimental 2.1. Materials FeSO4 ·7H2 O, FeCl3 ·6H2 O, oleic acid, toluene, tetrahydrofuran (THF) and 1,2-dichlorobenzene (DCB) were purchased from Sigma–Aldrich and used as received. Azobis(isobutyronitrile) (AIBN; Acros 98%) was recrystallized from methanol twice and stored at −20 ◦ C. Acetonitrile (ACN, Aldrich) and dimethylformamide (DMF, Aldrich) were distilled under vacuum after drying with CuSO4 . 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, 98%), N-hydroxylsuccinimide (NHS, 98%), bovine serum albumin (BSA), phosphate-buffered saline (PBS) tablets, kanamycin sulfate, neomycin sulfate, gentamycin sulfate, streptomycin sulfate, 2-mercaptobenzothiazole (MBT), 11-mercaptoundecanoic acid (MUA), HAuCl4 ·3H2 O and AgNO3 were purchased from Aldrich and used without further purification. The primary anti-kanamycin antibody was purchased from Beijing Wanger Biotechnology Co. Ltd (Beijing, China). N-acryloyl-l-glutamic acid (NAGA) monomer was synthesized according to published procedure [28]. Deionized water (>18.3 M cm) was used in all experiments.
2.2. Apparatus Transmission electron microscopy (TEM) observations of nanoparticles were conducted on JEOL JEM 1400 electron microscope at an acceleration voltage of 120 kV. High resolution TEM (HRTEM) observations and energy dispersive X-ray spectroscopy (EDX) analysis of bimetallic nanoparticles were conducted on JEOL JEM 2100F operating at an accelerating voltage of 200 kV. The sample for TEM observations was prepared by placing a 10 L nanoparticle solution on copper grids successively coated with thin films of carbon. No staining was applied. X-ray photoelectron spectroscopy (XPS) measurements were recorded on a SPECS XPS spectrometer equipped with a Mg K␣ X-ray source. After peak fitting of the C 1s spectra, all the spectra were calibrated in reference to the aliphatic C 1s component at a binding energy of 285.0 eV. Magnetic measurements were carried out at room temperature using a by using a physical properties measurement system (PPMS) from Quantum Design. UV–vis spectra were recorded on a Shimadzu UV–vis spectrometer UV-1601 with Shimadzu/UV Probe software. DeltaNu Examiner Raman microscope (DeltaNu Inc., Laramie, WY) with a 785 nm laser source, a motorized microscope stage sample holder, and a CCD detector was used to detect of kanamycin. During the measurements, a 20× objective was used and the laser spot diameter was 30 m. Samples were measured with 140 mW laser power, for 10 s acquisition time. Baseline correction was performed for all measurements. 2.3. Preparation of oleic acid-stabilized MNPs Oleic acid-stabilized MNPs were synthesized according to previously published protocol [29]. Briefly, 1.20 g of FeSO4 ·7H2 O and 2.05 g of FeCl3 ·6H2 O were dissolved in 50 mL of deionized water. After addition of 12.5 mL of NH4 OH (25%, v/v), the solution was stirred at room temperature for 30 min. Then, 0.5 mL of oleic acid was added into the black solution at 80 ◦ C and stirred for 1 h. 25 mL of nanoparticle solution and 25 mL of toluene were mixed in an extractor. By adding small amount of sodium chloride, MNPs were transferred into toluene phase. Finally, the nanoparticles were collected with a permanent magnet and dried under vacuum. 2.4. Preparation of BCPA-modified MNPs 2-[(Butylsulfanylcarbonylthiolsulfanyl) propionic acid] (BCPA) was prepared according to literature procedure [30]. BCPAmodified MNPs were prepared by a simple ligand exchange reaction of BCPA with oleic acid at the surface of MNPs. 100 mg of oleic acid-stabilized MNPs were dispersed into 1,2-dichlorobenzene (15 mL). After addition of BCPA (0.65 g, 2.73 mmol), the solution was stirred at 70 ◦ C for 24 h under nitrogen protection. Then, the reaction mixture was cooled to room temperature and the nanoparticles were collected with a magnet. After repeatedly washing of nanoparticles with dichlorobenzene and dichloromethane, BCPAmodified MNPs were dried under vacuum. 2.5. Surface-mediated RAFT polymerization A mixture of NAGA (30 mmol), AIBN (0.2 mmol) and BCPAmodified MNPs (100 mg) was dispersed in 5 mL of DMF/H2 O (4:1 v/v) and sonicated for 15 min. The solution was purged with nitrogen for 15 min and flame sealed. The ampoule was immersed in a preheated oil bath at 60 ◦ C and stirred for 36 h. Afterwards, the ampoule was rapid cooled by immersing it into water-ice bath. The mixture was diluted with THF and centrifuged at 5000 rpm for 3 min to collect magnetic nanoparticles. The dilution and centrifugation steps were repeated five times to remove ungrafted polymers originated from AIBN initiated polymerization. After
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
35
Scheme 1. Schematic representation of the synthesis of hybrid MNPs.
surface-mediated RAFT polymerization, the trithiocarbonate end groups were also removed via AIBN due to their toxicity and potential aminolysis reaction [31]. The hybrid MNPs were then collected with a magnet and washed with DMF for several times and then dried under vacuum.
2.6. Immobilization of anti-kanamycin Free carboxylic acids of poly(NAGA) were activated with EDC/NHS prior to immobilization of anti-kanamycin. 10 mg of the hybrid MNPs was dispersed in PBS (pH 7.4) solution containing 0.1 M EDC and 0.05 M NHS for 2 h at room temperature. The resultant particles were collected with a magnet and washed with PBS and water for several times. To covalent immobilization antikanamycin, the activated hybrid MNPs were dispersed in PBS buffer (pH 7.4) containing 20 g mL−1 anti-kanamycin. The reaction was allowed to continue for 2 h at room temperature under shaking. Then, the nanoparticles were washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the nanoparticles were dispersed in PBS buffer solution and stored in a refrigerator prior to use.
2.7. Preparation of Raman tags with anti-kanamycin antibody Au@Ag nanoparticles were synthesized as reported procedure [32]. After purification, the bimetallic nanoparticles were functionalized with 10 mM MUA and 1 mM MBT (Raman reporter) in absolute ethanol for overnight. After formation of SAM, the free carboxyl acid groups of MUA were activated with 0.05 M NHS and 0.1 M EDC for 2 h. The nanoparticles were separated with centrifugation and washed twice with PBS buffer (pH 7.4) solution. The nanoparticle solution (10 mL) was mixed with anti-kanamycin (PBS buffer, 20 g mL−1 ) for 2 h at room temperature under shaking. Then, the nanoparticles were washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the nanoparticles were dispersed in PBS buffer solution and stored in a refrigerator prior to use.
2.8. Preparation of sandwiched assay The anti-kanamycin immobilized MNPs were immersed in PBS solution containing different amounts of kanamycin (2 pg mL−1 –80 ng mL−1 ) for 30 min at room temperature under shaking. After, the nanoparticles were isolated with a magnet and washed with PBS solution containing 0.1 M NaCl. They were further reacted with Raman tags functionalized with anti-kanamycin described above in PBS solution at for 30 min room temperature under shaking. The resultant sandwich complex was collected with a magnet and washed with several times PBS solution containing 0.1 M NaCl and stored in refrigerator until SERS measurements. 3. Results and discussion Scheme 1 shows the procedure for creating hybrid MNPs coated with poly(NAGA) that bind anti-kanamycin molecules. The first step is the synthesis of BCPA-coated Fe3 O4 nanoparticles (Fe3 O4 @BCPA) with uniform size and shape via ligand exchange using OA-coated Fe3 O4 (Fe3 O4 @OA). The Fe3 O4 @OA nanoparticles were prepared via chemical co-precipitation method [28]. They were produced with a spherical morphology and an average diameter of 14 ± 6 nm (accounted on 140 nanoparticles, Fig. 1a). As shown in Fig. 1b, the size and shape of magnetite nanoparticles did not significantly change after the surface modification with BCPA. To demonstrate the successful BCPA ligand exchange onto Fe3 O4 nanoparticles, we inspected the nanoparticles using XPS (Fig. 2). The peaks at approximately 54, 92, 285, 530, and 710 eV which are attributed to Fe 3p, Fe 3s, C 1s, O 1s, and Fe 2p, respectively, are observed in wide scan XPS spectrum of the Fe3 O4 @OA nanoparticles (Fig 2a). Apart from these peaks, there appear new signals at approximately 162 and 225 eV assignable to S 2p and S 2s, respectively, in the wide scan XPS spectrum of the Fe3 O4 nanoparticles after ligand exchange with BCPA (Fig. 2b). The Fe3 O4 @BCPA nanoparticles were then used as a RAFT agent for the surface-mediated RAFT polymerization of NAGA, where the polymer chains were grafted directly from the nanoparticle surface to give magnetic and reactive core–shell hybrid nanoparticles (Scheme 1). The polymer shell has an average thickness of 22 ± 4 nm (Fig. 1c). It is also worthwhile to remark that the hybrid MNPs are
36
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 1. TEM images of (a) Fe3 O4 @OA and (b) Fe3 O4 @BCPA and (c) hybrid nanoparticles.
relatively uniformly distributed throughout the particles. Although the nanoparticles are aggregated, they exhibit relative concentric core–shell structures. This is expected because the polymeric shell restricts somehow the movement of the hybrid MNPs preventing the formation of eccentric structures [33].
In the wide scan XPS spectrum of the hybrid MNPs (Fig. 2c), the disappearance of Fe peaks and the appearance of N 1s peak at 400.2 eV also confirmed the formation of a thick polymeric layer on the Fe3 O4 (the maximum sampling depth of the XPS technique is ∼10 nm) [34]. The core level XPS spectra of the hybrid MNPs
Fig. 2. Survey-scan XPS spectra of (a) Fe3 O4 @OA, (b) Fe3 O4 @BCPA, survey scan and O1s, N1s and C1s core-level spectra of (c, d) hybrid nanoparticles.
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
37
Fig. 4. UV–vis spectra of (a) pure Au nanoparticles and (b) Au@Ag core–shell nanoparticles (inset). Images of pure Au and Au@Ag nanoparticle solutions. Fig. 3. Magnetic hysteresis curves of Fe3 O4 @OA (a) Fe3 O4 @BCPA (b), and hybrid nanoparticles (c).
consists of O 1s, N 1s and C 1s peaks curve fitted to components with binding energies of approximately 532.6 eV (C O), and 531.1 eV (C O) for O 1s, 400.1 eV (C N) for N 1s and 288.7 eV (C O), 285.8 eV (C O/C S/C N), and 284.8 eV (C C/C H) for C 1s (Fig. 2d). In order to study the magnetic behavior of the nanoparticles, magnetization experiments were performed. As shown in Fig. 3, the saturation magnetization was found to be about 61.8 emu g−1
and 72.0 emu g−1 for Fe3 O4 @OA and Fe3 O4 @BCPA, respectively. The higher magnetization found after BCPA ligand exchange was due to the lower weight of BCPA compare with OA. However, after the encapsulation of the magnetic nanoparticles inside the polymeric particle their magnetic properties drastically change. The magnetization versus magnetic field curve of the hybrid MNPs illustrates a dramatic decrease in saturation magnetization to 16.1 emu g−1 as a consequence of the polymeric shell. Nevertheless, in spite of the changes in the magnetic properties, the hybrid MNPs can be
Fig. 5. Representative TEM image (a) particle size distribution (b) and EDX spectrum of Au@Ag core–shell bimetallic nanoparticles (c).
38
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 6. SERS spectra of MBT labeled Au nanoparticles and MBT labeled Au@Ag nanoparticles.
extracted and recovered from the media using a magnetic field. Moreover, this level of saturation magnetization is deemed sufficient for biological applications [35,36]. After surface-mediated RAFT polymerization, the trithiocarbonate end groups were removed via AIBN due to their toxicity and potential aminolysis reaction [31,37]. To covalent immobilization of anti-kanamycin, free carboxylic acid groups of poly(NAGA) were first activated with EDC/NHS and then reacted with anti-kanamycin. Finally, the target kanamycin molecules were captured through the anti-kanamycin functionalized hybrid MNPs (Scheme 1). For sandwich assay applications, Au@Ag core–shell nanoparticles were also synthesized by growing silver nanoshells onto pre-synthesized citrate stabilized Au nanoparticles (∼14 nm) that served as nucleation sites. The UV–vis extinction spectra of monometalic citrate stabilized Au nanoparticles showed a band at 521 nm (Fig. 4a). For bimetallic core–shell nanoparticles, the gold band blue shifted and a plasmon band appeared at 410 nm (Fig. 4b) due to plasmon resonance of Ag shell. In addition, the color of the solution changed from pink to yellow with depositing Ag shell. HRTEM image of Au@Ag core–shell nanoparticle revealed two distinct contrasts: the dark gold core and the lighter silver shell (Fig. 5a). In the synthesis procedure, the particle aggregation was not observed and the average size distribution was 32 ± 2 nm. The silver shell was also uniform and the shell thickness was ∼9 nm. Importantly, there was no evidence for the formation of separate Ag nanoparticles in any TEM images. >Moreover, the EDX spectrum revealed presence of both gold and silver signals in the bimetallic nanoparticles (Fig. 5c). In order to compare the SERS activity of Au nanoparticles and Au@Ag nanoparticles, the same amount of SERS reporter (10 L of 0.01 M MBT) was added to an equal volume (1.0 mL) of these nanoparticle solutions with the same concentration under stirring to react overnight at room temperature. After washing away the unbound molecules, their SERS spectra were detected and given in Fig. 6. The strong bands of intensity appearing at 1014, 1139, 1248, 1394 and 1578 cm−1 in the SERS spectra may be assigned to the C C C bending, N C O bending, C N C bending, C C stretching and C H stretching vibrations [38]. The weak bands of intensity appearing at 607 and 712 cm−1 in the SERS spectra may be assigned to the C S stretching vibrations. MBT can exist in two tautomeric forms, the thione form with NH and thiol form with SH group. Generally, the lone-pair electron and aromatic system were easily bound to a metal surface [38]. In this case, these band intensities
can shift to the longer wavelengths and can be different from the band intensity of the bulk MBT. >Besides, the SERS intensity of silver coated nanoparticles is much higher than that of uncoated gold nanoparticles, indicating of the excellent SERS enhancement effect of silver coating, which can be explained in light of the ligand effect of core–shell nanoparticles [39]. Therefore, considering that the much stronger SERS signal should be less interfered by background noises, it can be inferred that Au@Ag nanoparticles would have an advantage over gold nanoparticles in the sandwich assay applications. The surface density of Au@Ag nanoparticles was optimized to achieve maximal adsorption performance of anti-kanamycin. For this purpose, the MUA/MBT mixed self-assembled monolayers (SAMs) were prepared by coadsorption from a mixed solution containing MUA and MBT. The mol ratio of MUA to MBT was varied from 0 to 100 mol%. Due to the competition between the two organic components, the concentration of MUA in the assembly solution determines the surface density of anti-kanamycin; i.e., low MUA concentrations lead to high anti-kanamycin densities. Terminal carboxylic acid groups of the mixed SAM are not as ordered as the homogeneous SAMs. Disorder may result either from phase separation or from full integration of two different components. With increased disorder, the terminal carboxylic acid groups are more accessible for the immobilization of anti-kanamycin. Meanwhile, the immobilization of anti-kanamycin onto Au@Ag nanoparticles also involves the formation of NHS ester with the carboxylic acid terminated SAMs using the water-soluble EDC. Side-chain amino groups of anti-kanamycin displace the terminal NHS groups, resulting in covalent immobilization of the protein. To determine the anti-kanamycin density that would immobilize the maximum amount of kanamycin, a series of mixed SAMs was prepared and adsorption of anti-kanamycin was measured by UV–vis based on Bradford protein assay [40]. As shown in Fig. 7a, there were no MUA molecules on the nanoparticles, a little amount of anti-kanamycin is adsorbed because non-specific interactions especially hydrophobic interactions occurred between MBT and anti-kanamycin. Once the MUA/MBT molar ratio reached to 10, there is sufficient MUA on the surface to immobilize nearly a monolayer of anti-kanamycin. As the MUA concentration increased further, the MUA density also increases, until eventually, the MUA molecules are packed so tight that anti-kanamycin cannot penetrate the MUA SAM and immobilize. As a result, less anti-kanamycin molecules can be immobilized. To maximize anti-kanamycin coverage, all subsequent mixed SAMs on Au@Ag nanoparticles were prepared with a molar ratio 10:1 of MUA/MBT. On the other hand, the adsorption capacity of Au@Ag nanoparticles is also dependent on the anti-kanamycin concentration. Therefore, we suspended the Au@Ag nanoparticles in the solution containing different anti-kanamycin concentrations. It can be detected that the adsorption capacity of Au@Ag nanoparticles gradually increased with anti-kanamycin concentrations (Fig. 7b). The adsorption capacity reached saturation as the concentration was 20 g mL−1 . The anti-kanamycin functionalized hybrid MNPs and antikanamycin modified Au@Ag nanoparticles were then bound each other via kanamycin molecules (Scheme 2). The SERS measurements were performed by using aggregated nanoparticles. The anti-kanamycin functionalized hybrid MNPs are not SERS active and therefore do not form hot spots with the anti-kanamycin modified Au@Ag nanoparticles. However, there will likely be plasmonic coupling between adjacent the anti-kanamycin modified Au@Ag nanoparticles. When the captured kanamycin molecules were labeled with anti-kanamycin modified Au@Ag nanoparticles, a sharp peak at 1248 cm−1 and peaks at 1394 and 1578 cm−1 appeared because of the MBT Raman signal. Therefore, the resultant SERS spectrum involves only MBT labeled SERS probe. In this case, the binding of kanamycin molecules to anti-kanamycins
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
39
Fig. 7. (a) Optimization of the surface composition for maximum anti-kanamycin binding (anti-kanamycin concentration: 20 g mL−1 ), (b) optimization of the optimal anti-kanamycin immobilized on the mixed SAM (10/1 molar ratio of MUA/MBT) on Au@Ag nanoparticles.
causes a plasmon and the peak at 1248 cm−1 of MBT increases depending on the concentration of kanamycin. Fig. 8a illustrates the SERS spectra of the sandwich assay for various concentrations of kanamycin. The concentration of kanamycin was varied from 2 pg mL−1 to 80 ng mL−1 . The Raman peak of MBT centered at 1248 cm−1 was used for the quantitative evaluation of kanamycin. In the absence of kanamycin, a weak SERS signal was observed (blank). This indicates that a small amount of anti-kanamycin modified Au@Ag nanoparticles still remained in the solution as a result of non-specific interaction with anti-kanamycin immobilized hybrid MNPs, even though most of the anti-kanamycin modified Au@Ag nanoparticles were removed from the solution by washing process. When kanamycin was added, the SERS signals were obviously enhanced with the increase in its concentration. Here,
the limit of detection was estimated to be 2 pg mL−1 because the Raman peak at 1248 cm−1 for this concentration is difficult to distinguish from that in the blank spectrum. The calibration curve, constructed from the peak intensity at 1248 cm−1 is shown in Fig. 8b, where the error bars indicate standard deviations from four measurements. The peak intensity gradually increases logarithmically with increasing concentration of kanamycin in the range from 2 pg mL−1 to 80 ng mL−1 . The inset figure shows a very good linear response in the lower concentration range from 2 to 80 pg mL−1 . Meanwhile, the anti-kanamycin functionalized hybrid MNPs were also exposed to the 2 ng mL−1 of kanamycin solution containing 100 ng mL−1 interfering substances (neomycin, gentamycin and streptomycin) to determine their selectivity for the kanamycin over the other molecules. The results were shown in Fig. 9a. The SERS
Scheme 2. Representative presentation of the preparation of sandwich assay.
40
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 8. (a) SERS spectra of the sandwich complex at different kanamycin concentrations. (b) Dose–response curve of the above kanamycin assay (y = 20519.3 − 1688.9x, R2 = 0.979, n = 4).
Fig. 9. SERS intensity at 1248 cm−1 of (a) anti-kanamycin functionalized hybrid MNPs exposed to neomycin (2 ng mL−1 ), gentamycin (2 ng mL−1 ), streptomycin (2 ng mL−1 ) and kanamycin (2 ng mL−1 ), and a kanamycin solution (2 ng mL−1 ) with equal amount (100 ng mL−1 ) of interfering substances, (b) regeneration of anti-kanamycin functionalized hybrid MNPs to kanamycin.
intensity variation due to the interference was less than 4% of intensity response in the absence of interferences, indicating that the selectivity of the anti-kanamycin functionalized hybrid MNPs was acceptable. To test the possibility of regenerating the anti-kanamycin functionalized hybrid MNPs, 0.2 M glycine-HCl (pH 2.0) was used to wash the nanoparticles. After the detection of 2.0 ng mL−1 kanamycin, the kanamycin immobilized hybrid MNPs was immersed into the glycine-HCl for 1 min to break the
anti-kanamycin–kanamycin linkage, and then used to detect 2.0 ng mL−1 kanamycin again. After four regeneration cycles (Fig. 9b), the kanamycin immobilized hybrid MNPs retained about 96% of their original value, and a relative standard deviations (RSD) of 3.1% was obtained. Although the prepared anti-kanamycin functionalized hybrid MNPs shows good selectively towards kanamycin, they are worth exploring the analytical utility of biosensor for a practical application. Detection of kanamycin in milks is of considerable interest,
Fig. 10. Kanamycin concentration-response curve for milk samples spiked with kanamycin in the range 2 pg mL−1 –80 ng mL−1 (y = 17742.7 − 1459.4x, R2 = 0.977, n = 4).
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41 Table 1 Recovery of kanamycin from spiked milk sample. Concentration of kanamycin in spiked milk sample (pg mL−1 )
Amount found (pg mL−1 )a
4.0 10 20 30 50 60
3.95 10.6 19.8 29.4 49.9 59.7
a b
± ± ± ± ± ±
0.15 0.27 0.31 0.41 0.65 0.85
Recovery (%)
RSD (%)b
98.7 106 99.0 98.0 99.8 99.5
3.89 2.54 1.57 1.39 1.30 1.42
Average value from four determinations for each concentration. Relative standard deviation of band intensity (RSD (%) = (SD/mean) × 100).
since milk is one of the most heavily regulated products in food industry because of the risk of having veterinary medicine residue [41]. The milk sample was firstly filtered through a sterile Millipore membrane (0.20 m) [36,37] and diluted five times with phosphate buffer solution. Moreover, kanamycin standard solution was spiked into the diluted milk, making the final concentrations of between 2 pg mL−1 and 80 ng mL−1 kanamycin, and then the experiments were carried out according to the aforementioned optimized conditions for kanamycin detection with the anti-kanamycin functionalized hybrid MNPs. The results were given in Fig. 10. When the calibration graphs obtained from kanamycin solutions in PBS (Fig. 8b) and kanamycin spiked milk samples were compared, it was found that the calibration curves were both in similar trend and the limit detection of kanamycin in the milk sample was determined to be 2.1 pg mL−1 , which was comparable or lower than the previously reported values [42–45]. Meanwhile, there is none zero intercept as the regression equation shown in Fig. 10. This indicates that there are non-specific interactions between the anti-kanamycin immobilized hybrid MNPs and the anti-kanamycin modified Au@Ag nanoparticles in the absence of kanamycin [46–48]. >Recovery was determined for milk samples spiked with 4–60 pg mL−1 kanamycin and was found to be between 98.0% and 106.0% (Table 1). Moreover, the RSD values changed between 1.30% and 3.89%, which clearly indicated the successful application of the developed SERS-based sandwich assay for detection of kanamycin in milk sample. Furthermore, the analysis time was less than 10 min, including washing and optical detection steps. 4. Conclusions In the present study, a new SERS-based sandwich assay was developed for extremely sensitive detection of kanamycin using a combination of anti-kanamycin immobilized hybrid MNPs and MBT labeled Au@Ag corel–shell nanoparticles as the recognition and SERS substrate, respectively, with an ability to be collected using a magnetic field. The LOD of the SERS-based sandwich assay was found to be 2 pg mL−1 , which was much lower than the previously reported results. The SERS-based sandwich assay showed good selectivity to kanamycin and long-term stability up to three months. In addition, this novel sandwich assay was successfully applied for kanamycin detection in milk sample. Therefore, it could be a promising tool for fast and reliable food analysis. Acknowledgment The authors thank Prof. Z. Suludere for TEM measurements. References [1] V. Manyanga, R. Dhulipalla, J. Hoogmartens, A. Erwin, J. Chromatogr. A 1217 (2010) 3748–3753.
41
[2] R. Oertel, V. Neumeister, W. Kirch, J. Chromatogr. A 1058 (2004) 197–201. [3] Y. Zhu, P. Chandra, K. Song, C. Ban, Y. Shim, Biosens. Bioelectron. 36 (2012) 29–34. [4] C. Chen, W. Zhai, D. Lu, W. Zheng, Mater. Res. Bull. 46 (2011) 583–587. [5] Y. Zhou, W. Yang, L. Zhang, Z. Wang, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 1603–1615. [6] Q. Wei, Y. Zhao, B. Du, D. Wu, H. Li, M. Yang, Food Chem. 134 (2012) 1601–1606. [7] S. Yu, Q. Wei, B. Du, H. Li, L. Yan, H. Ma, Y. Zhang, Biosens. Bioelectron. 48 (2013) 224–229. [8] E. Kaale, A. Schepdael, E. Roets, J. Hoogmartens, Electrophoresis 24 (2003) 1119–1125. [9] T. Kitagawa, K. Fujiwara, S. Tomonoh, K. Takahashi, M. Koida, J. Biochem. 94 (1983) 1165–1172. [10] J.D. Place, S.G. Thomson, J.F. Burd, C. Molinaro, R.A. Ott, F.C. Jensen, Hybridoma 3 (1984) 187–193. [11] P. Schnappinger, E. Usleber, E. Maltbauer, G. Terplan, Food Agric. Immunol. 5 (1993) 67–73. [12] J. Ara, Z. Gans, R. Sweeney, B. Wolf, J. Clin. Lab. Anal. 9 (1995) 320–324. [13] S.R. Raz, M.E.G.G. Bremer, W. Haasnoot, W. Norde, Anal. Chem. 81 (2009) 7743–7749. [14] M. Fransconi, R. Tel-Vered, M. Riskin, I. Willner, Anal. Chem. 82 (2010) 2512–2519. [15] R. Althaus, M.I. Berrugab, A. Monteroc, M. Rocac, M.P. Molinac, Anal. Chim. Acta 632 (2009) 156–162. [16] H.Y. Guo, L.H. Lu, C. Wu, J.G. Pan, J.W. Hu, Acta Chim. Sinica 67 (2009) 1603–1608. [17] C.G. Wang, Y. Chen, T.T. Wang, Z.F. Ma, Z.M. Su, Adv. Funct. Mater. 18 (2008) 355–361. [18] C.J. Orendorff, L. Gearheart, N.R. Jana, C. Murphy, J. Phys. Chem. Chem. Phys. 8 (2006) 165–170. [19] S. Lee, S. Kim, J. Choo, S.Y. Shin, Y.H. Lee, H.Y. Choi, S.H. Ha, K.H. Kang, C.H. Oh, Anal. Chem. 79 (2007) 916–922. [20] P.R. Sajanlal, T. Pradeep, Nano Res. 2 (2009) 306–320. [21] S. Pande, S.K. Ghosh, S. Praharaj, S. Basu, S. Jana, A. Pal, T. Tsukuda, T. Pal, J. Phys. Chem. C 111 (2007) 108806–110813. [22] Y.Y. Ma, W.Y. Li, E.C. Cho, Z.Y. Li, T.K. Yu, J. Zeng, Z.X. Xie, Y.N. Xia, ACS Nano 4 (2010) 6725–6734. [23] T.A. Taton, C.A. Mirkin, R.L. Letsinger, Science 289 (2000) 1757–1760. [24] Y. Cui, B. Ren, J.L. Yao, R.A. Gu, Z.Q. Tian, J. Phys. Chem. B 110 (2006) 4002–4006. [25] D. Mott, N.T.B. Thuy, Y. Aoki, S. Maenosono, Phil. Trans. R. Soc. A 368 (2010) 4275–4292. [26] Y. Ma, W. Li, E.C. Cho, Z. Li, T. Yu, J. Zeng, Z. Xie, Y. Xia, ACS Nano 6 (2010) 6725–6734. [27] B. Rodriguez-Gonzales, A. Burrows, M. Watanabe, C.J. Kiely, L.M.L. Marzan, J. Mater. Chem. 15 (2005) 1755–1759. [28] A. Winston, D.V.P.R. Varaprasad, J.J. Metterville, H. Rosenkrantz, J. Pharmacol. Exp. Ther. 232 (1985) 644–649. [29] Y. Sun, X. Ding, Z. Zheng, X. Cheng, X. Hu, Y. Peng, Chem. Commun. 26 (2006) 2765–2767. [30] C.J. Ferguson, R.J. Hughes, D. Nguyen, B.T.T. Pham, R.G. Gilbert, A.K. Serelis, C.H. Such, B.S. Hawkett, Macromolecules 38 (2005) 2191–2204. [31] A.W. York, F. Huang, C.L. McCormick, Biomacromolecules 11 (2010) 505–514. [32] L. Lu, H. Wang, Y. Zhou, S. Xi, H. Zhang, J. Hu, B. Zhao, Chem. Commun. (2002) 144–145. [33] J.P. Ge, Y.X. Hu, T.R. Zhang, Y.D. Yin, J. Am. Chem. Soc. 129 (2007) 8974–8975. [34] A. Zengin, T. Caykara, J. Polym. Sci. A: Polym. Chem. 50 (2012) 4443–4450. [35] C. Xu, K. Xu, H. Gu, R. Zheng, H. Liu, X. Zhang, Z. Guo, B. Xu, J. Am. Chem. Soc. 126 (2004) 9938–9939. [36] J.W.M. Bulte, M. Cuyper, D. Despres, J.A. Frank, J. Magn. Magn. Mater. 194 (1999) 204–209. [37] A. Zengin, U. Tamer, T. Caykara, Biomacromolecules 14 (2013) 3001–3009. [38] C.J. Lee, S.Y. Lee, M.R. Karim, M.S. Lee, Spectrochim. Acta A Mol. Biomol. Spectrosc. 68 (2007) 1313–1319. [39] S. Pande, S.K. Ghosh, S. Praharaj, S. Panigrahi, S. Basu, S. Jana, A. Pal, T. Tsukuda, T. Pal, J. Phys. Chem. C 111 (2007) 10806–10813. [40] J.-L. Gong, Y. Huang, J.-W. Chen, J.-H. Jiang, G.-L. Shen, R.-Q. Yu, Biosens. Bioelectron. 22 (2007) 1501–1507. [41] H.-J. Neubert, Anal. Chem. 78 (2006), 7908-7908. [42] S.R. Raz, M.G.E.G. Bremer, W. Haasnoot, W. Norde, Anal. Chem. 81 (2009) 7743–7749. [43] C.-Z. Yu, Y.-Z. He, G.-H. Fu, H.-Y. Xie, W.-E. Gan, J. Chromatogr. B 877 (2009) 333–338. [44] E.E.M.G. Loomans, J. Wiltenburg, M. Koets, A. Amerongen, J. Agric. Food. Chem. 51 (2003) 587–593. [45] S. Yu, Q. Wei, B. Du, D. Wu, H. Li, L. Yan, H. Ma, Y. Zhang, Biosens. Bioelectron. 48 (2013) 224–229. [46] H. Chon, S. Lee, S.W. Son, C.H. Oh, J. Choo, Anal. Chem. 81 (2010) 3029–3034. [47] M. Li, S.K. Cushing, H. Liang, S. Suri, D. Ma, N. Wu, Anal. Chem. 85 (2013) 2072–2078. [48] K. Chen, H. Han, Z. Luo, Y. Wang, X. Wang, Biosens. Bioelectron. 34 (2010) 118–124.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles Adem Zengin a,b , Ugur Tamer b , Tuncer Caykara a,∗ a b
Gazi University, Faculty of Science, Department of Chemistry, Besevler, 06500 Ankara, Turkey Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, Etiler, 06330 Ankara, Turkey
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Kanamycin immobilized hybrid magnetic nanoparticles were fabricated.
• They were sandwiched with the SERS substrate.
• The limit of detection was determined to be 2 pg mL−1 .
a r t i c l e
i n f o
Article history: Received 12 October 2013 Received in revised form 9 January 2014 Accepted 19 January 2014 Available online 30 January 2014 Keywords: Superparamagnetic nanoparticles Polymer brushes Kanamycin Surface enhanced Raman spectroscopy
a b s t r a c t Herein, we report the development of extremely sensitive sandwich assay of kanamycin using a combination of anti-kanamycin functionalized hybrid magnetic (Fe3 O4 ) nanoparticles (MNPs) and 2-mercaptobenzothiazole labeled Au-core@Ag-shell nanoparticles as the recognition and surfaceenhanced Raman scattering (SERS) substrate, respectively. The hybrid MNPs were first prepared via surface-mediated RAFT polymerization of N-acryloyl-l-glutamic acid in the presence of 2(butylsulfanylcarbonylthiolsulfanyl) propionic acid-modified MNPs as a RAFT agent and then biofunctionalized with anti-kanamycin, which are both specific for kanamycin and can be collected via a simple magnet. After separating kanamycin from the sample matrix, they were sandwiched with the SERS substrate. According to our experimental results, the limit of detection (LOD) was determined to be 2 pg mL−1 , this value being about 3–7 times more than sensitive than the LOD of previously reported results, which can be explained by the higher SERS activity of silver coated gold nanoparticles. The analysis time took less than 10 min, including washing and optical detection steps. Furthermore, the sandwich assay was evaluated for investigating the kanamycin specificity on neomycin, gentamycin and streptomycin and detecting kanamycin in artificially contaminated milk. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Kanamycin is an aminoglycoside antibiotic, which is fabricated by the fermentation of Streptomyces kanamyceticus and utilized to treat wide variety of infections by inducing mistranslation
∗ Corresponding author. Tel.: +90 312 202 11 24; fax: +90 312 212 22 79. E-mail address: [email protected] (T. Caykara). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.042
and indirectly inhibiting translocation during protein synthesis [1]. Similar to other aminoglycosides, kanamycin shows comparatively narrow safety margin and may cause many side effects such as loss of hearing, toxicity to kidneys, and allergic reactions to drugs [2] Moreover, the residual amount of kanamycin in the foodstuff may lead to antibiotic resistance from the pathogenic bacterial strains, which can endanger the consumer [3]. Hence, it is critical to developed sufficiently sensitive methods to detect kanamycin residue for clinical diagnosis and food safety. Recently,
34
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
a number of analytical methods, such as high performance liquid chromatography [4,5], label-free electrochemical immunosensor [6,7], capillary electrophoresis [8], enzyme-linked immunosorbent assay [9–12], surface plasmon resonance [13,14] and the microbiological multi-residue system [15] have been reported for the detection of kanamycin. However, most of those above-mentioned methods are often time consuming, tedious, and require great amount of reagents, solvents, and expensive apparatus. Thus, it is desirable to develop a low cost, selective and sensitive method for kanamycin detection with less reagent consumption. Recently, SERS-based assay method using antibody conjugated metal nanoparticles has attracted great interest from many researchers because of its rapid end sensitive sensing capability. >Moreover, most of them have focused on the application of Au nanoparticles SERS probes to assay systems [16,17]. Although Au nanoparticles are stable, biocompatible, they only exhibit a moderate enhancement in SERS detection compared to Ag nanoparticles [18]. However, the application of those Ag nanoparticles is more or less limited on account of their easily aggregations in solutions [19]. Therefore, bimetallic Au@Ag or Ag@Au core–shell nanoparticles have become the focus of attention due to their greater SERS activity in comparison with individual Ag and Au nanoparticles [20–25]. In practical terms, the Ag@Au structure has shown many drawbacks, high suppression of the optical properties of Ag by the Au shell, as well as significant challenges in preparing a probe with uniform structure [24,25]. In many cases, the relatively high reduction potential of Au causes Ag to be oxidized in the coating process, leading to structures with hollow cores or formation of non-continuous Au shell [25–27]. They are also inherently unstable due to the exposure of Ag to the outside environment, making them non-ideal for biomolecular sensing and diagnostics. However, the Au@Ag nanoparticle system is still very attractive as a platform for sensing applications. In this report, we present an extremely sensitive detection method for kanamycin utilizing anti-kanamycin functionalized hybrid MNPs and 2-mercaptobenzothiazole (MBT) labeled Au@Ag core–shell nanoparticles as probe and SERS substrate, respectively. Furthermore, this method was also tested for the kanamycin specificity on neomycin, gentamycin and streptomycin, and for the detection of kanamycin in artificially contaminated milk. The results undoubtedly demonstrated the superiority of SERS-based sandwich assay with extremely increased sensitivity using bimetallic Au@Ag core–shell nanoparticles.
2. Experimental 2.1. Materials FeSO4 ·7H2 O, FeCl3 ·6H2 O, oleic acid, toluene, tetrahydrofuran (THF) and 1,2-dichlorobenzene (DCB) were purchased from Sigma–Aldrich and used as received. Azobis(isobutyronitrile) (AIBN; Acros 98%) was recrystallized from methanol twice and stored at −20 ◦ C. Acetonitrile (ACN, Aldrich) and dimethylformamide (DMF, Aldrich) were distilled under vacuum after drying with CuSO4 . 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, 98%), N-hydroxylsuccinimide (NHS, 98%), bovine serum albumin (BSA), phosphate-buffered saline (PBS) tablets, kanamycin sulfate, neomycin sulfate, gentamycin sulfate, streptomycin sulfate, 2-mercaptobenzothiazole (MBT), 11-mercaptoundecanoic acid (MUA), HAuCl4 ·3H2 O and AgNO3 were purchased from Aldrich and used without further purification. The primary anti-kanamycin antibody was purchased from Beijing Wanger Biotechnology Co. Ltd (Beijing, China). N-acryloyl-l-glutamic acid (NAGA) monomer was synthesized according to published procedure [28]. Deionized water (>18.3 M cm) was used in all experiments.
2.2. Apparatus Transmission electron microscopy (TEM) observations of nanoparticles were conducted on JEOL JEM 1400 electron microscope at an acceleration voltage of 120 kV. High resolution TEM (HRTEM) observations and energy dispersive X-ray spectroscopy (EDX) analysis of bimetallic nanoparticles were conducted on JEOL JEM 2100F operating at an accelerating voltage of 200 kV. The sample for TEM observations was prepared by placing a 10 L nanoparticle solution on copper grids successively coated with thin films of carbon. No staining was applied. X-ray photoelectron spectroscopy (XPS) measurements were recorded on a SPECS XPS spectrometer equipped with a Mg K␣ X-ray source. After peak fitting of the C 1s spectra, all the spectra were calibrated in reference to the aliphatic C 1s component at a binding energy of 285.0 eV. Magnetic measurements were carried out at room temperature using a by using a physical properties measurement system (PPMS) from Quantum Design. UV–vis spectra were recorded on a Shimadzu UV–vis spectrometer UV-1601 with Shimadzu/UV Probe software. DeltaNu Examiner Raman microscope (DeltaNu Inc., Laramie, WY) with a 785 nm laser source, a motorized microscope stage sample holder, and a CCD detector was used to detect of kanamycin. During the measurements, a 20× objective was used and the laser spot diameter was 30 m. Samples were measured with 140 mW laser power, for 10 s acquisition time. Baseline correction was performed for all measurements. 2.3. Preparation of oleic acid-stabilized MNPs Oleic acid-stabilized MNPs were synthesized according to previously published protocol [29]. Briefly, 1.20 g of FeSO4 ·7H2 O and 2.05 g of FeCl3 ·6H2 O were dissolved in 50 mL of deionized water. After addition of 12.5 mL of NH4 OH (25%, v/v), the solution was stirred at room temperature for 30 min. Then, 0.5 mL of oleic acid was added into the black solution at 80 ◦ C and stirred for 1 h. 25 mL of nanoparticle solution and 25 mL of toluene were mixed in an extractor. By adding small amount of sodium chloride, MNPs were transferred into toluene phase. Finally, the nanoparticles were collected with a permanent magnet and dried under vacuum. 2.4. Preparation of BCPA-modified MNPs 2-[(Butylsulfanylcarbonylthiolsulfanyl) propionic acid] (BCPA) was prepared according to literature procedure [30]. BCPAmodified MNPs were prepared by a simple ligand exchange reaction of BCPA with oleic acid at the surface of MNPs. 100 mg of oleic acid-stabilized MNPs were dispersed into 1,2-dichlorobenzene (15 mL). After addition of BCPA (0.65 g, 2.73 mmol), the solution was stirred at 70 ◦ C for 24 h under nitrogen protection. Then, the reaction mixture was cooled to room temperature and the nanoparticles were collected with a magnet. After repeatedly washing of nanoparticles with dichlorobenzene and dichloromethane, BCPAmodified MNPs were dried under vacuum. 2.5. Surface-mediated RAFT polymerization A mixture of NAGA (30 mmol), AIBN (0.2 mmol) and BCPAmodified MNPs (100 mg) was dispersed in 5 mL of DMF/H2 O (4:1 v/v) and sonicated for 15 min. The solution was purged with nitrogen for 15 min and flame sealed. The ampoule was immersed in a preheated oil bath at 60 ◦ C and stirred for 36 h. Afterwards, the ampoule was rapid cooled by immersing it into water-ice bath. The mixture was diluted with THF and centrifuged at 5000 rpm for 3 min to collect magnetic nanoparticles. The dilution and centrifugation steps were repeated five times to remove ungrafted polymers originated from AIBN initiated polymerization. After
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
35
Scheme 1. Schematic representation of the synthesis of hybrid MNPs.
surface-mediated RAFT polymerization, the trithiocarbonate end groups were also removed via AIBN due to their toxicity and potential aminolysis reaction [31]. The hybrid MNPs were then collected with a magnet and washed with DMF for several times and then dried under vacuum.
2.6. Immobilization of anti-kanamycin Free carboxylic acids of poly(NAGA) were activated with EDC/NHS prior to immobilization of anti-kanamycin. 10 mg of the hybrid MNPs was dispersed in PBS (pH 7.4) solution containing 0.1 M EDC and 0.05 M NHS for 2 h at room temperature. The resultant particles were collected with a magnet and washed with PBS and water for several times. To covalent immobilization antikanamycin, the activated hybrid MNPs were dispersed in PBS buffer (pH 7.4) containing 20 g mL−1 anti-kanamycin. The reaction was allowed to continue for 2 h at room temperature under shaking. Then, the nanoparticles were washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the nanoparticles were dispersed in PBS buffer solution and stored in a refrigerator prior to use.
2.7. Preparation of Raman tags with anti-kanamycin antibody Au@Ag nanoparticles were synthesized as reported procedure [32]. After purification, the bimetallic nanoparticles were functionalized with 10 mM MUA and 1 mM MBT (Raman reporter) in absolute ethanol for overnight. After formation of SAM, the free carboxyl acid groups of MUA were activated with 0.05 M NHS and 0.1 M EDC for 2 h. The nanoparticles were separated with centrifugation and washed twice with PBS buffer (pH 7.4) solution. The nanoparticle solution (10 mL) was mixed with anti-kanamycin (PBS buffer, 20 g mL−1 ) for 2 h at room temperature under shaking. Then, the nanoparticles were washed with PBS solution containing 0.1 M NaCl and 1% BSA for several times. After washing procedure, the nanoparticles were dispersed in PBS buffer solution and stored in a refrigerator prior to use.
2.8. Preparation of sandwiched assay The anti-kanamycin immobilized MNPs were immersed in PBS solution containing different amounts of kanamycin (2 pg mL−1 –80 ng mL−1 ) for 30 min at room temperature under shaking. After, the nanoparticles were isolated with a magnet and washed with PBS solution containing 0.1 M NaCl. They were further reacted with Raman tags functionalized with anti-kanamycin described above in PBS solution at for 30 min room temperature under shaking. The resultant sandwich complex was collected with a magnet and washed with several times PBS solution containing 0.1 M NaCl and stored in refrigerator until SERS measurements. 3. Results and discussion Scheme 1 shows the procedure for creating hybrid MNPs coated with poly(NAGA) that bind anti-kanamycin molecules. The first step is the synthesis of BCPA-coated Fe3 O4 nanoparticles (Fe3 O4 @BCPA) with uniform size and shape via ligand exchange using OA-coated Fe3 O4 (Fe3 O4 @OA). The Fe3 O4 @OA nanoparticles were prepared via chemical co-precipitation method [28]. They were produced with a spherical morphology and an average diameter of 14 ± 6 nm (accounted on 140 nanoparticles, Fig. 1a). As shown in Fig. 1b, the size and shape of magnetite nanoparticles did not significantly change after the surface modification with BCPA. To demonstrate the successful BCPA ligand exchange onto Fe3 O4 nanoparticles, we inspected the nanoparticles using XPS (Fig. 2). The peaks at approximately 54, 92, 285, 530, and 710 eV which are attributed to Fe 3p, Fe 3s, C 1s, O 1s, and Fe 2p, respectively, are observed in wide scan XPS spectrum of the Fe3 O4 @OA nanoparticles (Fig 2a). Apart from these peaks, there appear new signals at approximately 162 and 225 eV assignable to S 2p and S 2s, respectively, in the wide scan XPS spectrum of the Fe3 O4 nanoparticles after ligand exchange with BCPA (Fig. 2b). The Fe3 O4 @BCPA nanoparticles were then used as a RAFT agent for the surface-mediated RAFT polymerization of NAGA, where the polymer chains were grafted directly from the nanoparticle surface to give magnetic and reactive core–shell hybrid nanoparticles (Scheme 1). The polymer shell has an average thickness of 22 ± 4 nm (Fig. 1c). It is also worthwhile to remark that the hybrid MNPs are
36
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 1. TEM images of (a) Fe3 O4 @OA and (b) Fe3 O4 @BCPA and (c) hybrid nanoparticles.
relatively uniformly distributed throughout the particles. Although the nanoparticles are aggregated, they exhibit relative concentric core–shell structures. This is expected because the polymeric shell restricts somehow the movement of the hybrid MNPs preventing the formation of eccentric structures [33].
In the wide scan XPS spectrum of the hybrid MNPs (Fig. 2c), the disappearance of Fe peaks and the appearance of N 1s peak at 400.2 eV also confirmed the formation of a thick polymeric layer on the Fe3 O4 (the maximum sampling depth of the XPS technique is ∼10 nm) [34]. The core level XPS spectra of the hybrid MNPs
Fig. 2. Survey-scan XPS spectra of (a) Fe3 O4 @OA, (b) Fe3 O4 @BCPA, survey scan and O1s, N1s and C1s core-level spectra of (c, d) hybrid nanoparticles.
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
37
Fig. 4. UV–vis spectra of (a) pure Au nanoparticles and (b) Au@Ag core–shell nanoparticles (inset). Images of pure Au and Au@Ag nanoparticle solutions. Fig. 3. Magnetic hysteresis curves of Fe3 O4 @OA (a) Fe3 O4 @BCPA (b), and hybrid nanoparticles (c).
consists of O 1s, N 1s and C 1s peaks curve fitted to components with binding energies of approximately 532.6 eV (C O), and 531.1 eV (C O) for O 1s, 400.1 eV (C N) for N 1s and 288.7 eV (C O), 285.8 eV (C O/C S/C N), and 284.8 eV (C C/C H) for C 1s (Fig. 2d). In order to study the magnetic behavior of the nanoparticles, magnetization experiments were performed. As shown in Fig. 3, the saturation magnetization was found to be about 61.8 emu g−1
and 72.0 emu g−1 for Fe3 O4 @OA and Fe3 O4 @BCPA, respectively. The higher magnetization found after BCPA ligand exchange was due to the lower weight of BCPA compare with OA. However, after the encapsulation of the magnetic nanoparticles inside the polymeric particle their magnetic properties drastically change. The magnetization versus magnetic field curve of the hybrid MNPs illustrates a dramatic decrease in saturation magnetization to 16.1 emu g−1 as a consequence of the polymeric shell. Nevertheless, in spite of the changes in the magnetic properties, the hybrid MNPs can be
Fig. 5. Representative TEM image (a) particle size distribution (b) and EDX spectrum of Au@Ag core–shell bimetallic nanoparticles (c).
38
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 6. SERS spectra of MBT labeled Au nanoparticles and MBT labeled Au@Ag nanoparticles.
extracted and recovered from the media using a magnetic field. Moreover, this level of saturation magnetization is deemed sufficient for biological applications [35,36]. After surface-mediated RAFT polymerization, the trithiocarbonate end groups were removed via AIBN due to their toxicity and potential aminolysis reaction [31,37]. To covalent immobilization of anti-kanamycin, free carboxylic acid groups of poly(NAGA) were first activated with EDC/NHS and then reacted with anti-kanamycin. Finally, the target kanamycin molecules were captured through the anti-kanamycin functionalized hybrid MNPs (Scheme 1). For sandwich assay applications, Au@Ag core–shell nanoparticles were also synthesized by growing silver nanoshells onto pre-synthesized citrate stabilized Au nanoparticles (∼14 nm) that served as nucleation sites. The UV–vis extinction spectra of monometalic citrate stabilized Au nanoparticles showed a band at 521 nm (Fig. 4a). For bimetallic core–shell nanoparticles, the gold band blue shifted and a plasmon band appeared at 410 nm (Fig. 4b) due to plasmon resonance of Ag shell. In addition, the color of the solution changed from pink to yellow with depositing Ag shell. HRTEM image of Au@Ag core–shell nanoparticle revealed two distinct contrasts: the dark gold core and the lighter silver shell (Fig. 5a). In the synthesis procedure, the particle aggregation was not observed and the average size distribution was 32 ± 2 nm. The silver shell was also uniform and the shell thickness was ∼9 nm. Importantly, there was no evidence for the formation of separate Ag nanoparticles in any TEM images. >Moreover, the EDX spectrum revealed presence of both gold and silver signals in the bimetallic nanoparticles (Fig. 5c). In order to compare the SERS activity of Au nanoparticles and Au@Ag nanoparticles, the same amount of SERS reporter (10 L of 0.01 M MBT) was added to an equal volume (1.0 mL) of these nanoparticle solutions with the same concentration under stirring to react overnight at room temperature. After washing away the unbound molecules, their SERS spectra were detected and given in Fig. 6. The strong bands of intensity appearing at 1014, 1139, 1248, 1394 and 1578 cm−1 in the SERS spectra may be assigned to the C C C bending, N C O bending, C N C bending, C C stretching and C H stretching vibrations [38]. The weak bands of intensity appearing at 607 and 712 cm−1 in the SERS spectra may be assigned to the C S stretching vibrations. MBT can exist in two tautomeric forms, the thione form with NH and thiol form with SH group. Generally, the lone-pair electron and aromatic system were easily bound to a metal surface [38]. In this case, these band intensities
can shift to the longer wavelengths and can be different from the band intensity of the bulk MBT. >Besides, the SERS intensity of silver coated nanoparticles is much higher than that of uncoated gold nanoparticles, indicating of the excellent SERS enhancement effect of silver coating, which can be explained in light of the ligand effect of core–shell nanoparticles [39]. Therefore, considering that the much stronger SERS signal should be less interfered by background noises, it can be inferred that Au@Ag nanoparticles would have an advantage over gold nanoparticles in the sandwich assay applications. The surface density of Au@Ag nanoparticles was optimized to achieve maximal adsorption performance of anti-kanamycin. For this purpose, the MUA/MBT mixed self-assembled monolayers (SAMs) were prepared by coadsorption from a mixed solution containing MUA and MBT. The mol ratio of MUA to MBT was varied from 0 to 100 mol%. Due to the competition between the two organic components, the concentration of MUA in the assembly solution determines the surface density of anti-kanamycin; i.e., low MUA concentrations lead to high anti-kanamycin densities. Terminal carboxylic acid groups of the mixed SAM are not as ordered as the homogeneous SAMs. Disorder may result either from phase separation or from full integration of two different components. With increased disorder, the terminal carboxylic acid groups are more accessible for the immobilization of anti-kanamycin. Meanwhile, the immobilization of anti-kanamycin onto Au@Ag nanoparticles also involves the formation of NHS ester with the carboxylic acid terminated SAMs using the water-soluble EDC. Side-chain amino groups of anti-kanamycin displace the terminal NHS groups, resulting in covalent immobilization of the protein. To determine the anti-kanamycin density that would immobilize the maximum amount of kanamycin, a series of mixed SAMs was prepared and adsorption of anti-kanamycin was measured by UV–vis based on Bradford protein assay [40]. As shown in Fig. 7a, there were no MUA molecules on the nanoparticles, a little amount of anti-kanamycin is adsorbed because non-specific interactions especially hydrophobic interactions occurred between MBT and anti-kanamycin. Once the MUA/MBT molar ratio reached to 10, there is sufficient MUA on the surface to immobilize nearly a monolayer of anti-kanamycin. As the MUA concentration increased further, the MUA density also increases, until eventually, the MUA molecules are packed so tight that anti-kanamycin cannot penetrate the MUA SAM and immobilize. As a result, less anti-kanamycin molecules can be immobilized. To maximize anti-kanamycin coverage, all subsequent mixed SAMs on Au@Ag nanoparticles were prepared with a molar ratio 10:1 of MUA/MBT. On the other hand, the adsorption capacity of Au@Ag nanoparticles is also dependent on the anti-kanamycin concentration. Therefore, we suspended the Au@Ag nanoparticles in the solution containing different anti-kanamycin concentrations. It can be detected that the adsorption capacity of Au@Ag nanoparticles gradually increased with anti-kanamycin concentrations (Fig. 7b). The adsorption capacity reached saturation as the concentration was 20 g mL−1 . The anti-kanamycin functionalized hybrid MNPs and antikanamycin modified Au@Ag nanoparticles were then bound each other via kanamycin molecules (Scheme 2). The SERS measurements were performed by using aggregated nanoparticles. The anti-kanamycin functionalized hybrid MNPs are not SERS active and therefore do not form hot spots with the anti-kanamycin modified Au@Ag nanoparticles. However, there will likely be plasmonic coupling between adjacent the anti-kanamycin modified Au@Ag nanoparticles. When the captured kanamycin molecules were labeled with anti-kanamycin modified Au@Ag nanoparticles, a sharp peak at 1248 cm−1 and peaks at 1394 and 1578 cm−1 appeared because of the MBT Raman signal. Therefore, the resultant SERS spectrum involves only MBT labeled SERS probe. In this case, the binding of kanamycin molecules to anti-kanamycins
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
39
Fig. 7. (a) Optimization of the surface composition for maximum anti-kanamycin binding (anti-kanamycin concentration: 20 g mL−1 ), (b) optimization of the optimal anti-kanamycin immobilized on the mixed SAM (10/1 molar ratio of MUA/MBT) on Au@Ag nanoparticles.
causes a plasmon and the peak at 1248 cm−1 of MBT increases depending on the concentration of kanamycin. Fig. 8a illustrates the SERS spectra of the sandwich assay for various concentrations of kanamycin. The concentration of kanamycin was varied from 2 pg mL−1 to 80 ng mL−1 . The Raman peak of MBT centered at 1248 cm−1 was used for the quantitative evaluation of kanamycin. In the absence of kanamycin, a weak SERS signal was observed (blank). This indicates that a small amount of anti-kanamycin modified Au@Ag nanoparticles still remained in the solution as a result of non-specific interaction with anti-kanamycin immobilized hybrid MNPs, even though most of the anti-kanamycin modified Au@Ag nanoparticles were removed from the solution by washing process. When kanamycin was added, the SERS signals were obviously enhanced with the increase in its concentration. Here,
the limit of detection was estimated to be 2 pg mL−1 because the Raman peak at 1248 cm−1 for this concentration is difficult to distinguish from that in the blank spectrum. The calibration curve, constructed from the peak intensity at 1248 cm−1 is shown in Fig. 8b, where the error bars indicate standard deviations from four measurements. The peak intensity gradually increases logarithmically with increasing concentration of kanamycin in the range from 2 pg mL−1 to 80 ng mL−1 . The inset figure shows a very good linear response in the lower concentration range from 2 to 80 pg mL−1 . Meanwhile, the anti-kanamycin functionalized hybrid MNPs were also exposed to the 2 ng mL−1 of kanamycin solution containing 100 ng mL−1 interfering substances (neomycin, gentamycin and streptomycin) to determine their selectivity for the kanamycin over the other molecules. The results were shown in Fig. 9a. The SERS
Scheme 2. Representative presentation of the preparation of sandwich assay.
40
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41
Fig. 8. (a) SERS spectra of the sandwich complex at different kanamycin concentrations. (b) Dose–response curve of the above kanamycin assay (y = 20519.3 − 1688.9x, R2 = 0.979, n = 4).
Fig. 9. SERS intensity at 1248 cm−1 of (a) anti-kanamycin functionalized hybrid MNPs exposed to neomycin (2 ng mL−1 ), gentamycin (2 ng mL−1 ), streptomycin (2 ng mL−1 ) and kanamycin (2 ng mL−1 ), and a kanamycin solution (2 ng mL−1 ) with equal amount (100 ng mL−1 ) of interfering substances, (b) regeneration of anti-kanamycin functionalized hybrid MNPs to kanamycin.
intensity variation due to the interference was less than 4% of intensity response in the absence of interferences, indicating that the selectivity of the anti-kanamycin functionalized hybrid MNPs was acceptable. To test the possibility of regenerating the anti-kanamycin functionalized hybrid MNPs, 0.2 M glycine-HCl (pH 2.0) was used to wash the nanoparticles. After the detection of 2.0 ng mL−1 kanamycin, the kanamycin immobilized hybrid MNPs was immersed into the glycine-HCl for 1 min to break the
anti-kanamycin–kanamycin linkage, and then used to detect 2.0 ng mL−1 kanamycin again. After four regeneration cycles (Fig. 9b), the kanamycin immobilized hybrid MNPs retained about 96% of their original value, and a relative standard deviations (RSD) of 3.1% was obtained. Although the prepared anti-kanamycin functionalized hybrid MNPs shows good selectively towards kanamycin, they are worth exploring the analytical utility of biosensor for a practical application. Detection of kanamycin in milks is of considerable interest,
Fig. 10. Kanamycin concentration-response curve for milk samples spiked with kanamycin in the range 2 pg mL−1 –80 ng mL−1 (y = 17742.7 − 1459.4x, R2 = 0.977, n = 4).
A. Zengin et al. / Analytica Chimica Acta 817 (2014) 33–41 Table 1 Recovery of kanamycin from spiked milk sample. Concentration of kanamycin in spiked milk sample (pg mL−1 )
Amount found (pg mL−1 )a
4.0 10 20 30 50 60
3.95 10.6 19.8 29.4 49.9 59.7
a b
± ± ± ± ± ±
0.15 0.27 0.31 0.41 0.65 0.85
Recovery (%)
RSD (%)b
98.7 106 99.0 98.0 99.8 99.5
3.89 2.54 1.57 1.39 1.30 1.42
Average value from four determinations for each concentration. Relative standard deviation of band intensity (RSD (%) = (SD/mean) × 100).
since milk is one of the most heavily regulated products in food industry because of the risk of having veterinary medicine residue [41]. The milk sample was firstly filtered through a sterile Millipore membrane (0.20 m) [36,37] and diluted five times with phosphate buffer solution. Moreover, kanamycin standard solution was spiked into the diluted milk, making the final concentrations of between 2 pg mL−1 and 80 ng mL−1 kanamycin, and then the experiments were carried out according to the aforementioned optimized conditions for kanamycin detection with the anti-kanamycin functionalized hybrid MNPs. The results were given in Fig. 10. When the calibration graphs obtained from kanamycin solutions in PBS (Fig. 8b) and kanamycin spiked milk samples were compared, it was found that the calibration curves were both in similar trend and the limit detection of kanamycin in the milk sample was determined to be 2.1 pg mL−1 , which was comparable or lower than the previously reported values [42–45]. Meanwhile, there is none zero intercept as the regression equation shown in Fig. 10. This indicates that there are non-specific interactions between the anti-kanamycin immobilized hybrid MNPs and the anti-kanamycin modified Au@Ag nanoparticles in the absence of kanamycin [46–48]. >Recovery was determined for milk samples spiked with 4–60 pg mL−1 kanamycin and was found to be between 98.0% and 106.0% (Table 1). Moreover, the RSD values changed between 1.30% and 3.89%, which clearly indicated the successful application of the developed SERS-based sandwich assay for detection of kanamycin in milk sample. Furthermore, the analysis time was less than 10 min, including washing and optical detection steps. 4. Conclusions In the present study, a new SERS-based sandwich assay was developed for extremely sensitive detection of kanamycin using a combination of anti-kanamycin immobilized hybrid MNPs and MBT labeled Au@Ag corel–shell nanoparticles as the recognition and SERS substrate, respectively, with an ability to be collected using a magnetic field. The LOD of the SERS-based sandwich assay was found to be 2 pg mL−1 , which was much lower than the previously reported results. The SERS-based sandwich assay showed good selectivity to kanamycin and long-term stability up to three months. In addition, this novel sandwich assay was successfully applied for kanamycin detection in milk sample. Therefore, it could be a promising tool for fast and reliable food analysis. Acknowledgment The authors thank Prof. Z. Suludere for TEM measurements. References [1] V. Manyanga, R. Dhulipalla, J. Hoogmartens, A. Erwin, J. Chromatogr. A 1217 (2010) 3748–3753.
41
[2] R. Oertel, V. Neumeister, W. Kirch, J. Chromatogr. A 1058 (2004) 197–201. [3] Y. Zhu, P. Chandra, K. Song, C. Ban, Y. Shim, Biosens. Bioelectron. 36 (2012) 29–34. [4] C. Chen, W. Zhai, D. Lu, W. Zheng, Mater. Res. Bull. 46 (2011) 583–587. [5] Y. Zhou, W. Yang, L. Zhang, Z. Wang, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 1603–1615. [6] Q. Wei, Y. Zhao, B. Du, D. Wu, H. Li, M. Yang, Food Chem. 134 (2012) 1601–1606. [7] S. Yu, Q. Wei, B. Du, H. Li, L. Yan, H. Ma, Y. Zhang, Biosens. Bioelectron. 48 (2013) 224–229. [8] E. Kaale, A. Schepdael, E. Roets, J. Hoogmartens, Electrophoresis 24 (2003) 1119–1125. [9] T. Kitagawa, K. Fujiwara, S. Tomonoh, K. Takahashi, M. Koida, J. Biochem. 94 (1983) 1165–1172. [10] J.D. Place, S.G. Thomson, J.F. Burd, C. Molinaro, R.A. Ott, F.C. Jensen, Hybridoma 3 (1984) 187–193. [11] P. Schnappinger, E. Usleber, E. Maltbauer, G. Terplan, Food Agric. Immunol. 5 (1993) 67–73. [12] J. Ara, Z. Gans, R. Sweeney, B. Wolf, J. Clin. Lab. Anal. 9 (1995) 320–324. [13] S.R. Raz, M.E.G.G. Bremer, W. Haasnoot, W. Norde, Anal. Chem. 81 (2009) 7743–7749. [14] M. Fransconi, R. Tel-Vered, M. Riskin, I. Willner, Anal. Chem. 82 (2010) 2512–2519. [15] R. Althaus, M.I. Berrugab, A. Monteroc, M. Rocac, M.P. Molinac, Anal. Chim. Acta 632 (2009) 156–162. [16] H.Y. Guo, L.H. Lu, C. Wu, J.G. Pan, J.W. Hu, Acta Chim. Sinica 67 (2009) 1603–1608. [17] C.G. Wang, Y. Chen, T.T. Wang, Z.F. Ma, Z.M. Su, Adv. Funct. Mater. 18 (2008) 355–361. [18] C.J. Orendorff, L. Gearheart, N.R. Jana, C. Murphy, J. Phys. Chem. Chem. Phys. 8 (2006) 165–170. [19] S. Lee, S. Kim, J. Choo, S.Y. Shin, Y.H. Lee, H.Y. Choi, S.H. Ha, K.H. Kang, C.H. Oh, Anal. Chem. 79 (2007) 916–922. [20] P.R. Sajanlal, T. Pradeep, Nano Res. 2 (2009) 306–320. [21] S. Pande, S.K. Ghosh, S. Praharaj, S. Basu, S. Jana, A. Pal, T. Tsukuda, T. Pal, J. Phys. Chem. C 111 (2007) 108806–110813. [22] Y.Y. Ma, W.Y. Li, E.C. Cho, Z.Y. Li, T.K. Yu, J. Zeng, Z.X. Xie, Y.N. Xia, ACS Nano 4 (2010) 6725–6734. [23] T.A. Taton, C.A. Mirkin, R.L. Letsinger, Science 289 (2000) 1757–1760. [24] Y. Cui, B. Ren, J.L. Yao, R.A. Gu, Z.Q. Tian, J. Phys. Chem. B 110 (2006) 4002–4006. [25] D. Mott, N.T.B. Thuy, Y. Aoki, S. Maenosono, Phil. Trans. R. Soc. A 368 (2010) 4275–4292. [26] Y. Ma, W. Li, E.C. Cho, Z. Li, T. Yu, J. Zeng, Z. Xie, Y. Xia, ACS Nano 6 (2010) 6725–6734. [27] B. Rodriguez-Gonzales, A. Burrows, M. Watanabe, C.J. Kiely, L.M.L. Marzan, J. Mater. Chem. 15 (2005) 1755–1759. [28] A. Winston, D.V.P.R. Varaprasad, J.J. Metterville, H. Rosenkrantz, J. Pharmacol. Exp. Ther. 232 (1985) 644–649. [29] Y. Sun, X. Ding, Z. Zheng, X. Cheng, X. Hu, Y. Peng, Chem. Commun. 26 (2006) 2765–2767. [30] C.J. Ferguson, R.J. Hughes, D. Nguyen, B.T.T. Pham, R.G. Gilbert, A.K. Serelis, C.H. Such, B.S. Hawkett, Macromolecules 38 (2005) 2191–2204. [31] A.W. York, F. Huang, C.L. McCormick, Biomacromolecules 11 (2010) 505–514. [32] L. Lu, H. Wang, Y. Zhou, S. Xi, H. Zhang, J. Hu, B. Zhao, Chem. Commun. (2002) 144–145. [33] J.P. Ge, Y.X. Hu, T.R. Zhang, Y.D. Yin, J. Am. Chem. Soc. 129 (2007) 8974–8975. [34] A. Zengin, T. Caykara, J. Polym. Sci. A: Polym. Chem. 50 (2012) 4443–4450. [35] C. Xu, K. Xu, H. Gu, R. Zheng, H. Liu, X. Zhang, Z. Guo, B. Xu, J. Am. Chem. Soc. 126 (2004) 9938–9939. [36] J.W.M. Bulte, M. Cuyper, D. Despres, J.A. Frank, J. Magn. Magn. Mater. 194 (1999) 204–209. [37] A. Zengin, U. Tamer, T. Caykara, Biomacromolecules 14 (2013) 3001–3009. [38] C.J. Lee, S.Y. Lee, M.R. Karim, M.S. Lee, Spectrochim. Acta A Mol. Biomol. Spectrosc. 68 (2007) 1313–1319. [39] S. Pande, S.K. Ghosh, S. Praharaj, S. Panigrahi, S. Basu, S. Jana, A. Pal, T. Tsukuda, T. Pal, J. Phys. Chem. C 111 (2007) 10806–10813. [40] J.-L. Gong, Y. Huang, J.-W. Chen, J.-H. Jiang, G.-L. Shen, R.-Q. Yu, Biosens. Bioelectron. 22 (2007) 1501–1507. [41] H.-J. Neubert, Anal. Chem. 78 (2006), 7908-7908. [42] S.R. Raz, M.G.E.G. Bremer, W. Haasnoot, W. Norde, Anal. Chem. 81 (2009) 7743–7749. [43] C.-Z. Yu, Y.-Z. He, G.-H. Fu, H.-Y. Xie, W.-E. Gan, J. Chromatogr. B 877 (2009) 333–338. [44] E.E.M.G. Loomans, J. Wiltenburg, M. Koets, A. Amerongen, J. Agric. Food. Chem. 51 (2003) 587–593. [45] S. Yu, Q. Wei, B. Du, D. Wu, H. Li, L. Yan, H. Ma, Y. Zhang, Biosens. Bioelectron. 48 (2013) 224–229. [46] H. Chon, S. Lee, S.W. Son, C.H. Oh, J. Choo, Anal. Chem. 81 (2010) 3029–3034. [47] M. Li, S.K. Cushing, H. Liang, S. Suri, D. Ma, N. Wu, Anal. Chem. 85 (2013) 2072–2078. [48] K. Chen, H. Han, Z. Luo, Y. Wang, X. Wang, Biosens. Bioelectron. 34 (2010) 118–124.
