Materials Science and Engineering C 74 (2017) 365–373 Contents lists available at ScienceDirect Materials Science and Engineering C j ourna l homepage: www.e lsev ie r .com/ locate /msec Synthesis of a new magnetic-MIP for the selective detection of 1-chloro-2,4-dinitrobenzene, a highly allergenic compound Rosario Josefina Uzuriaga-Sánchez a,c, Ademar Wong a, Sabir Khan a, Maria I. Pividori b, Gino Picasso c,⁎,1, Maria D.P.T. Sotomayor a,⁎,1 a Department of Analytical Chemistry, Institute of Chemistry, State University of São Paulo (UNESP), 14801-970 Araraquara, SP, Brazil b Sensors and Biosensors Group, Department of Chemistry, Autonomous University of Barcelona (UAB), 08193, Bellaterra, Barcelona, Spain c Laboratory of Physical Chemistry Research, Faculty of Science, National University of Engineering, Av. Tupac Amaru 210, Rimac, Lima, Peru ⁎ Corresponding authors. E-mail addresses: gpicasso@uni.edu.pe (G. Picasso), m (M.D.P.T. Sotomayor). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.msec.2016.12.019 0928-4931/© 2016 Elsevier B.V. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 24 September 2016 Received in revised form 29 November 2016 Accepted 5 December 2016 Available online 9 December 2016 Molecularly imprinted polymers (MIPs) in combination with magnetic nanoparticles, in a core@shell format, were studied for selective detection of 1-chloro-2,4-dinitrobenzene (CDNB), a powerful allergenic substance. Magnetic nanoparticles were prepared by the co-precipitation method andmixed with oleic acid (OA). This ma- terial was then encapsulated in three types of hydrophobic polymeric matrix, poly-(MA-co-EDGMA), poly-(AA- co-EDGMA), and poly-(1-VN-co-EDGMA), by the mini-emulsion method. These matrices were used due to their ability to interact specifically with the functional groups of the analyte. Finally, the MIP-CDNB was obtained on the magnetic-hydrophobic surfaces using precipitation polymerization in the presence of the analyte. XRD dif- fraction patterns suggested the presence ofmagnetite in the composite and SEM analysis revealed a nanoparticle size between 10 and 18 nm. Under the optimized adsorption conditions, the magnetic-MIP material showed a higher adsorption capacity (5.1mg g−1) than its non-magnetic counterpart (4.2mg g−1). In tests of the selectiv- ity of the magnetic-MIP towards CDNB,α-values of 2.5 and 10.4, respectively, were obtained for dichlorophenol and o-nitrophenol, two structurally similar compounds, and no adsorptionwas observed for any other non-anal- ogous analyte. Themagnetic-MIP andmagnetic-NIPwere applied usingwater enrichedwith 0.5mg L−1 of CDNB, achieving recovery values of 83.8(±0.8)% and 66(±1)%, respectively, revealing the suitability of thematerial for detection of CDNB. © 2016 Elsevier B.V. All rights reserved. Keywords: Biomimetic polymers Plastic antibodies Magnetic-MIP 1-Chloro-2,4-dinitrobenzene Environmental control Allergenic substances 1. Introduction 1-Chloro-2,4-dinitrobenzene (CDNB), a recognized substrate of the glutathione-S-transferase (GST) enzyme [1] and an irreversible inhibi- tor of human thioredoxin reductase, is a highly allergenic substance. In individuals exposed to it, this compound can cause effects ranging from simple skin irritation, which can lead to primary or allergic type dermatitis [2,3], as well as induction of type IV or delayed-type hyper- sensitivity (DTH) [4,5]. The DTH reactions are mediated by T-cells and monocytes/macrophages, rather than by antibodies. CDNB is used me- dicinally to assess T-cell activity in patients, providing a useful diagnos- tic test for immunocompromised patients, as well as to treat warts. In the most severe cases, abnormal T-cell function can result in opportu- nistic infections, including infection with mycobacteria, fungi, and pilar@iq.unesp.br parasites associatedwith syndromes includingmucocutaneous candidi- asis, tuberculin skin reactions, granulomatous inflammations (sarcoido- sis, Crohn's disease), and eye and respiratory irritations [6–10]. CDNB is a toxic xenobiotic that is known to cause oxidative stress and cell death [11]. Its effects on human health make it important to be able to detect and quantify this analyte. CDNB is used as an algaecide in the coolant water of air-conditioning systems [12], where there is an evidence that sensitization to the com- pound can occur following persistent exposure to low concentrations or by a single exposure to a high concentration. Such exposures can lead to allergic skin reactions or chronic poisoning [6]. CDNB is also used as a re- agent for the detection and determination of pyridine compounds and for the synthesis of many other organic compounds. In light of the ad- verse effects of CDNB on workers in the chemical industry and the pub- lic health sector, as well as in the environment, there is a need for methods capable of sensitive and reliable quantification of this chemical. CDNB is widely used in quantification of the glutathione-s-transfer- ase enzyme [13–16] and its substrates [17]. However, despite its envi- ronmental and health relevance, only very few reports have been http://crossmark.crossref.org/dialog/?doi=10.1016/j.msec.2016.12.019&domain=pdf http://dx.doi.org/10.1016/j.msec.2016.12.019 mailto:mpilar@iq.unesp.br http://dx.doi.org/10.1016/j.msec.2016.12.019 http://www.sciencedirect.com/science/journal/09284931 www.elsevier.com/locate/msec 366 R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 published concerning CDNB quantification [18]. The present work pro- poses a novel technique for the selective extraction and efficient preconcentration of CDNB, prior to its subsequent simple quantification using techniques such as UV/Vis spectrometry, HPLC, or any other con- ventional method. In recent years, our research group has worked on the development of highly efficient molecularly imprinted polymers (MIPs) for various analytes [19–22], in order to obtain selective and specific materials for the extraction of analytes present at very low concentrations [23–24]. These materials with artificially generated recognition sites are capable of rebinding a target molecule with high selectivity, due to the forma- tion of specific binding sites with shape and functionalities complemen- tary to the analyte [24], acting similarly to natural receptors with antibody-antigen interactions. For this reason, MIPs are also called plas- tic antibodies [25]. Once their synthesis has been optimized,MIPs can be prepared in a very simple and inexpensive way. In addition, they can operate in a wide range of solution pH, at high temperatures, in high ionic strength solutions, and in organic solvents [26–27]. We [28–30] and other research groups [31–35] have synthesized hybrid materials based on magnetic nanoparticles decorated with MIPs, which have shown high selectivity towards various analytes. The combination ofMIPs andmagnetic nanoparticles offersmany at- tractive and innovative possibilities in biomedicine and bioanalysis. Magnetic particles can be coated or modifiedwith biological molecules, enabling manipulation using an external magnetic field [36], and their uses can be extended to other areas, including the analysis of industrial effluents or environmental samples. Amongother advantages, these hy- brid materials offer enhanced selectivity, durability, and the possibility of reuse [28]. This work describes, for the first time, the preparation of magnetic nanoparticles modified with an MIP selective to CDNB, in a core@shell format (magnetic-MIP). In addition, a systematic study was undertaken to select the functional monomers for the intermediate organic poly- meric layer, which was prepared by the mini-emulsion method, before obtaining the magnetic-MIP. 2. Experimental 2.1. Reagents All the reagents used were analytical grade. 1-Chloro-2,4-dinitro- benzene (CDNB), ferrous sulfate (FeSO4·7H2O), ferric sulfate (Fe2(SO4)3 9H2O), oleic acid (OA, 9-octadecenoic acid, C18H34O2), sodi- um dodecyl sulfate (SDS), methyl methacrylate, ethylene glycol dimethacrylate (EGDMA), methacrylic acid (MA), acrylic acid (AA), 1- vinylimidazole (1-VN), potassium persulfate, bisphenol A (BPA), aceto- nitrile, methylene diphenyl diisocyanate-4 (MDI), and phloroglucinol were all purchased from Sigma-Aldrich. Methanol, ethanol, sulfuric acid, acetic acid, and sodium hydroxide (NaOH) were obtained from Synth. Solutions were prepared in electrochemical grade deionized water (resistivity ≥18 MΩ cm at 25 °C). 2.2. Preparation of supermagnetic magnetite–OA nanoparticles Supermagnetic nanoparticles of Fe3O4 were prepared according to the chemical co-precipitation method by adding a solution of NaOH (2.5% w/v) to a mixture of iron salts with Fe2+/Fe3+ molar ratio of 1:2, at 80 °C, maintaining a constant pH 9 until the process was com- plete [37]. Afterwards, the precipitatewas collected using a neodymium magnet (5 × 5 × 50 mm), filtered, and washed with deionized water. Themagnetite was aged for 24 h in a dry atmosphere andwas then vig- orouslymixedwith oleic acid in a ratio of 1:3 (w/w), in order to coat the magnetite with a single surfactant layer of OA (magnetite–OA nanopar- ticles). The magnetite–OA nanoparticles were washed with water and ethanol, followed by storage in a desiccator for 3 days to ensure com- plete removal of solvent [38]. 2.3. Encapsulation of magnetite-OA nanoparticles in a polymeric matrix: Influence of the functional monomer on MIP efficiency The magnetite-OA nanoparticles were first encapsulated in a poly- meric matrix, following the procedure described by Valero-Navarro et al. [39]. Evaluation was made of matrices based on three different hy- drophobic polymers: poly-(MA-co-EDGMA), poly-(AA-co-EDGMA), and poly-(1-VN-co-EGDMA). This approach was based on the different abilities of the functional monomers (FM) to interact with the function- al groups of the analyte. The hydrophobic polymerization was performed by the mini- emulsion method, using SDS as surfactant. A 2 g portion of magne- tite–OA was dispersed in 4 mL of n-hexane/chloroform (1:1, v/v) and then added to 450 mL of deionized water containing 337.5 mg of SDS. This mixture was maintained in an ice-water mixture for 10 min, followed by sonication for 20 min. The resulting emulsion was transferred slowly to a flask containing 1.5 mL of a mixture of the FM and EGDMA (40:60, w/w), under mechanical agitation. The mixture was stirred for 1 h, after which 180 mg of KPS (potassium persulfate) was added to begin the polymerization. The reaction sys- temwas heated at 65 °C under a N2 atmosphere for 24 h. The product was washed 6 times with deionized water, 5 times with acetone, and 5 times with chloroform, in order to eliminate undesirable moieties. The product, consisting of the hydrophobic FM/EGDMA polymer on the magnetic nanoparticles, was denoted magnetite-poly-(FM-co- EDGMA). 2.4. Synthesis of magnetic–MIP selective to CDNB The MIP selective to CDNB was synthesized over the magnetic-hy- drophobic surface using precipitation polymerization in the presence of the analyte, forming a core@shell structure (Fig. 1). In this procedure, 1-chloro-2,4-dinitrobenzene (0.20 mmol), bisphenol A (0.70 mmol), MDI (0.82 mmol), and phloroglucinol (0.33 mmol) were dissolved in a mixture of tetrahydrofuran (8 mL) and 16 mg of the magnetite-poly- (FM-co-EDGMA) (4.5% w/w). This procedure was carried out based on the reports of Dickert [40–41] and other research groups [39,42–43], in which the MIP was based on a polyurethane polymer imprinted with the analyte [41]. To obtain polyurethanes, the synthesis requires a polyol (bisphenol A - 2,2-bis(4-hydroxyphenyl)propane), a diisocyanate (MDI - methylene diphenyl diisocyanate-4), and a diol chain extender (phloroglucinol) [44]. These compounds were therefore used to obtain the MIP based on a polyurethane matrix, with phloroglucinol as an additional cross-linker [45] together with the EDGMA immobilized on the magnetite nanoparticles. The polymerization process was carried out under continuous me- chanical stirring for 2 days, in the dark at room temperature. After the polymerization, the material was washed with methanol/acetic acid (9:1, v/v) in a Soxhlet extractor and then dried under vacuum for 2 days. The NIP (non-imprinted polymer) used as a control polymer was prepared using the same procedure employed for the MIP, but without the analyte molecule. 2.5. Characterization of the magnetic-MIPs 2.5.1. X-ray diffraction (XRD) XRD analysis was performed using a Rigaku Miniflex II instrument operated using the following conditions: Cu Kα radiation (λ = 1.5418 Å), 15 mA, 30 kV, Ni filter, 2θ scanning range of 5–80°, step size of 0.10°, and step time of 2.5 s. The crystal phases were identified using diffraction data from JCPDS (International Centre for Diffraction Data). The mean crystallite sizes were estimated using the Debye- Scherrer equation and the selected peaks were fitted by a Gaussian function. 367R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 2.5.2. Determination of specific surface area and porosity The textural properties were studied by N2 sorption measurements at 77 K (liquid nitrogen temperature), using a Micromeritics Gemini VII 2390t instrument. Prior to the adsorption experiments, the samples were degassed under vacuumwith helium, at 80 °C, for 4 h. The specific surface area was calculated according to the BET method and the pore size distribution was evaluated using the BJH (Barrett-Joyner-Halenda) procedure. 2.5.3. Scanning electron microscopy (SEM) The particle size and surface morphology of the samples were ana- lyzed by field emission gun scanning electron microscopy (FEG-SEM), using a JEOL 7500F instrument. 2.5.4. Transmission electron microscopy (TEM) High-resolution transmission electron microscopy (HR-TEM) analy- ses were performed using a Philips CM200 instrument with resolution of 1.9 Å. 2.6. Quantification of CDNB by HPLC Chromatographic analyses were performed using a Shimadzu 20A liquid chromatograph equipped with an SPD-20A UV/Vis detector, an SIL-20A autosampler, and a DGU-20A5 degasser. Separation was achieved with a Phenomenex Luna C18 column (250 mm × 4.6 mm), and the chromatography system was controlled by a microcomputer. The analyses were performed in isocratic mode, with a mobile phase composed of a mixture of water:methanol (40:60, v/v), at a flow rate of 1.0 mL min−1, an injection volume of 20 μL, and a detector wave- length of 260 nm. CDNB standard solutions were prepared in methanol at concentrations of between 0.1 and 25 mg L−1. A typical chromato- gram and a typical analytical curve obtained under these conditions are shown in the Supplementary material (S1 and S2). Fig. 1. Schematic representation of the synthesis of the magnetic- 2.7. Binding and selective adsorption experiments The binding experiment was carried out by adding 5 mg of Mag- MIP and Mag-NIP to different glass vials, followed by addition (at pH ~6) of 3.0 mL of a solution of CDNB at different concentrations, obtained from a stock solution of the analyte dissolved in methanol. The mixture was submitted to rotary shaking for 120 min. The magnetic polymers were separated from the suspensions using a neodymium magnet and the remaining solutions were filtered through a 0.45 μm membrane prior to HPLC analysis (Section 2.6). The binding capacity of the samples (Qe) was calculated using the following eq. [28]: Qe ¼ Co−Ceð ÞV m ð1Þ where Qe (mg g−1) is the experimental equilibrium adsorption capacity, C0 (mg L−1) is the initial concentration of analyte, Ce (mg L−1) is the equilibrium concentration of analyte, V (mL) is the volume of solution, and m (g) is the weight of Mag-MIP or Mag-NIP. Determination of the selectivity towards CDNB employed competitive adsorption experiments performed using two types of analytes: compounds chemically analogous to CDNB (o-nitrophenol and 3,5-dichlorophenol) and compounds with different natures (4-(4- nitrophenylazo)resorcinol, p-dimethylaminoazobenzene, and caffeine). The distribution coefficient (Kd, mL g−1) and selectivity (α) were calculated using Eqs. (2) and (3), respectively [28]: Kd ¼ Qe Ce ð2Þ α ¼ Kd CDNBð Þ Kd interferentð Þ ð3Þ MIP for 1-chloro-2,4-dinitrobenzene, proposed in this work. Fig. 2. X-ray diffractograms of the sample prior to reaction with OA (M1) and of the magnetite-OA sample (M2). Fig. 4.N2 sorption isotherms ofmagnetite sampleswithout (M1) andwith oleic acid (M2). 368 R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 3. Results and discussion 3.1. Structural and morphological characterization of the magnetite The diffraction patterns of the nanoparticles based on magnetite showed excellent agreement with the characteristic peaks of the mag- netite standard (JCPDS file no 19-0629). Fig. 2 shows the diffractograms for the sample prior to reactionwith OA (M1) and for themagnetite-OA sample (M2). Peaks corresponding to the diffraction planes (220), (311), (400), (422), (511), and (440) of Fe3O4 can be seen at angles of 30.19°, 35.58°, 43.35°, 53.57°, 56.95°, and 62.70°, respectively, suggest- ing the presence of a magnetite-like structure as the main component in the composite. The XRD patterns indicated that the degree of crystallinity of the sample preparedwith surfactant (M2)was lower, compared to its coun- terpart preparedwithout OA (M1), as expected due to the anti-agglom- eration effect of the surfactant. The anti-agglomeration effect of oleic acid and the size of the parti- cleswere also analyzed by scanning electronmicroscopy (SEM). Sample M1 showed particles ranging from 20 to 90 nm in diameter, with the presence of agglomerations of different dimensions (Fig. 3a), while its counterpart sample M2 showed the formation of smaller particles with diameters from10 to 18 nm (Fig. 3b). These characteristics indicat- ed the advantageous use of OA in terms of the homogeneity and unifor- mity of shape of the magnetite particles. The surface areas (S) of the M1 and M2 samples were evaluated using the N2 sorption technique, with fitting of the isotherms according to the BET method. As shown in Fig. 4, both samples presented typical type IV isotherms ofmesoporous powders, with H1 andH3 type hyster- esis loops for M1 and M2, respectively. An important finding was that the BET specific surface area of sample M2was higher than that of sam- ple M1 (Table 1), which could lead to greater MIP formation on the M2 Fig. 3. Scanning electron micrographs of a) the sample without surfactant (M1), at a mag surface, hence increasing the number of selective cavities in the mag- netic-MIP. As expected, the use of oleic acid as a surfactant resulted in the formation of particles with smaller pore size and higher surface area in the magnetite-OA nanomaterial (Table 1). 3.2. Influence of the functional monomer in the hydrophobic polymeric ma- trix of poly-(FM-co-EDGMA) on the magnetic-MIP efficiency In order to identify appropriate conditions for the synthesis of mag- netic-MIP, one of themore important parameters, described for the first time in this work, was the interaction of the analyte (1–chloro–2,4–di- nitrobenzene) with the functional monomer used in preparation of the MIP. In the preparation of the hydrophobic polymeric matrix, the monomers were not directly used in the presence of the analyte. How- ever, their interaction was very important in the step prior to the MIP synthesis, because the stronger the interaction between the monomer and the solidmatrix, the greater the number of selective cavities expect- ed to be formed in the final magnetic-MIP material. In this study, selec- tion was made of three different monomers that have been used previously as functional monomers in MIP synthesis: 1-vinylimidazol (1-VN) [46], methacrylic acid (MA) [47], and acrylic acid (AA) [48]. Fig. 5 shows the efficiency of adsorption of CDNB for the magnetic- MIPs prepared using each of the three monomers. It can be seen from Fig. 5 that efficient imprinting of the magnetic- MIP using a simple monomer system had a strong influence on adsorp- tion of the template used to prepare the hydrophobic polymeric matrix. nification of 100,000×, and b) magnetite-OA (M2), at a magnification of 250,000×. Table 1 BET surface area (S) and pore diameter (dBET) of samples M1 and M2. Sample S (m2 g−1) dBET (nm) M1 48.8 ± 0.2 23.63 ± 0.08 M2 70.1 ± 0.3 16.99 ± 0.03 Fig. 6. Transmission electron micrographs of the magnetic-MIP for CDNB at two different magnifications (a and b). 369R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 The magnetic-MIP synthesized using poly-(MA-co-EDGMA) showed the best adsorption capacity (4.1 mg g−1), with lower values obtained for the magnetic-MIPs prepared using AA and 1-VN (2.0 and 0.9 mg g−1, respectively). This can be explained by the electron-with- drawing nature of the CDNBmolecule [49], which generates a strong in- teraction in the presence of methacrylic acid. The double bond in methacrylic acid and its methacrylate derivatives is highly polarized and its electronic density is decreased due to the electron-withdrawing and resonance effects induced by the carbonyl group [50]. Bothmethac- rylates and acrylates (with lower strength) therefore react readily with nucleophilic reagents such as CDNB. In the present case, a hypothetical interaction between the double bond in methacrylic acid and the nitro groups of the analyte could be considered as an electrostatic interaction. In the case of 1-VN, the high electron density caused repulsion of CDNB, which explained theworst results generatedusing thismonomer. Based on these results, subsequent experiments were performed using the magnetic-MIP prepared using methacrylic acid in the hydrophobic layer. 3.3. Morphological characterization of the magnetic-MIP for CDNB Fig. 6 shows TEMmicrographs of the magnetic-MIP at two different scales. The agglomeration ofmagnetite nanoparticles surrounded by the MIP layer can be seen in Fig. 6a. Fig. 6b shows successful formation of the double circle of magnetite@MIP microspheres, with the porous MIP shell uniformly coating the dark Fe3O4 core. Due to the small size of the magnetite-OA, a greater quantity of recognition sites were formed, so a higher analyte adsorption capacity was expected. Similar profiles were observed for the corresponding magnetic-NIP (Fig. 7). However, the material was less homogeneous and showed lower porosity (Table 2), due to the absence of selective cavities. Fig. 8 shows SEM images obtained for themagnetic-MIP at twomag- nifications. It is evident that the magnetic-MIP presented a homoge- neous appearance, with the presence of very well defined spherical microparticles with diameters of up to 900 nm (Fig. 8b). These micro- particles were of different and better shapes, compared to those previ- ously synthesized by us using the precipitation method [28]. This 0 10 20 30 0 2 4 6 poly-(MA-co-EDGMA) poly-(AA-co-EDGMA) poly-(1-VN-co-EDGMA) Q e (m g g-1 ) Ce (mg L-1) Fig. 5. Adsorption isotherms of the magnetic-MIPs synthesized with hydrophobic polymeric matrices of poly-(MA-co-EDGMA), poly-(AA-co-EDGMA), and poly-(1-VN-co- EDGMA). Measurements were carried out following the procedure described in Section 2.7. Fig. 7. Transmission electron micrographs of the magnetic-NIP for CDNB at two different magnifications (a and b). Table 2 BET surface area (S), pore diameter (dBET), and pore volume (VBJH) of the magnetic-MIP and magnetic-NIP samples. Sample S (m2 g−1) dBET (nm) VBJH (cm3 g−1) Magnetic-MIP 120.0 ± 0.6 9.92 ± 0.04 0.364 ± 0.001 Magnetic-NIP 75.0 ± 0.5 7.81 ± 0.02 0.212 ± 0.002 0 2 4 6 8 10 12 14 16 3.85 3.90 3.95 4.00 Q e ( m g g-1 ) pH Fig. 9. Influence of pH on adsorption of 1-chloro-2,4-dinitrobenzene from the extraction solution. Measurements were carried out as described in Section 2.7, with Canalyte = 10 mg L−1. 370 R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 revealed that the polymerization method used in this work provided improved synthesis of themagnetic-MIP, compared to other procedures described in the literature [28,42]. The N2-sorption technique was employed to estimate the surface area of the magnetic-MIP and the volume and dimensions of the pores. The surface area of the magnetic-MIP was greater than that of themagnetic-NIP (Table 2). In addition, the data shown in Table 2 indi- cated that the synthesizedmaterials possessedmesoporous characteris- tics, as expected for materials based on MIPs. The pore sizes of both materials were b10 nm, in contrast to the 24 nm pore size of the mag- netite nanoparticles (Table 1), indicating that theMIP layer was present on the surface of the magnetic material, in agreement with the TEM images. 3.4. Optimization of the adsorption capacity of the magnetic-MIP for CDNB The pH is a very important parameter in adsorption processes be- cause the acidity of the solution can affect the amount of the analyte adsorbed. The extraction of CDNB using the prepared magnetic-MIP was investigated using solutions with different pH values and the opti- mal adsorption of CDNB occurred at pH near to 6 (Fig. 9). This could be explained considering the pKa values of the analyte and the polyure- thane-MIP. It has been found previously that the chain extenders exert a very strong influence on the pH characteristics of polyurethanes [51]. In this case, the diol chain extender used was phloroglucinol, which has first and second pKa values of 8.0 and 9.2, respectively [52]. At pH between 3.0 and 9.0, the adsorption was almost constant (Fig. 9), indicative of favorable interaction between the analyte and the MIP. At these pH values the polyurethane-MIP was in protonated form and its interaction with the CDNB analyte was maximized, because in this pH range CDNB presents high electronic density from nitro and chloride groups, due to its pKa value of 1.8 [53]. This facilitated its trans- fer into the cavities of the MIP, which was highest at pH 6. The adsorp- tion was lower at pH above 9.0, at which the two species were negatively charged, introducing a repulsive effect that reduced the ana- lyte-MIP interaction. Hence, pH 6.0 was used in all the subsequent experiments. Once the optimal pH had been established, evaluation was made of the influence of the sorption time on the retention properties of the Fig. 8. Scanning electron micrographs of the ma MIP. The quantity of CDNB adsorbed by the magnetic-MIP increased with extraction time, up to a maximum at 120 min, after which the ad- sorption decreased slowly (Fig. 10). Hence, an adsorption time of 120 min was used in the remaining experiments. It is important to point out that the magnetic-MIP always adsorbed greater amounts of CDNB, compared to its corresponding magnetic-NIP counterpart, as shown in Fig. 10. This trendwas similar to the behavior of othermagnet- ic-MIPs prepared in our laboratory [29] and could be explained by the different surface areas of magnetic-MIP and magnetic-NIP (Table 2), due to the presence of the selective cavities in the imprinted polymer. An important point to note is that themagnetic-NIPwas useful as a con- trol to take account of the non-specific adsorption [30] of CDNB by the magnetic adsorbent material. 3.5. Selectivity studies Experiments to evaluate the selectivity of the prepared magnetic- MIP were performed for each interfering compound under the previ- ously optimized experimental conditions. Fig. 11 shows the isotherms obtained for two compounds with chemical structures analogous to CDNB, namely 3,5-dichlorophenol (curve b in Fig. 11) and o-nitrophe- nol (curve c in Fig. 11), compared to the analyte isotherm. The results shown in Fig. 11 enabled estimation of the selectivity of the magnetic- MIP to CDNB, relative to the interfering compounds. From Eqs. (2) and (3), α-values of 2.5 and 10.4 were obtained for 3,5-dichlorophenol gnetic-MIP at two different magnifications. 0 30 60 90 120 150 180 0 2 4 6 magnetic-MIP magnetic-NIP Q e ( m g g-1 ) Time (min) Fig. 10. Time-dependent adsorption of 1-chloro-2,4-dinitrobenzene. Measurements were carried out as is described in Section 2.7, with Canalyte = 10 mg L−1. Fig. 12. Chemical structures of (a) 1-chloro-2,4-dinitrobenzene, (b) 3,5-dichlorophenol, (c) o-nitrophenol, (d) 4-(4-nitrophenylazo)resorcinol, (e) caffeine, and (f) p- dimethylaminoazobenzene. 371R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 and o-nitrophenol, respectively. Hence, even in the presence of com- poundswith similar chemical structures (Fig. 12), the synthesizedmag- netic-MIP was highly selective to CDNB. For other compounds with different structures (Fig. 12), practically no adsorptionwas observed. The findings of thiswork therefore indicat- ed that the prepared magnetic-MIP showed a maximum adsorption ca- pacity in the presence of CDNB, due to the presence of nanocavitieswith selective sizes and the influence of specific sample-receptor interactions. 3.6. Analytical characteristics and application of the magnetic-MIP In order to apply the magnetic-MIP, the HPLC method (Supplemen- tary material S1 and S2) was optimized and showed a broad response range (0.1–25 mg L−1) together with LOD (0.03 mg L−1) and retention time (9.1 min) similar to values reported in the literature [18]. Evalua- tion was then made of the efficiency of the magnetic materials (MIP and NIP) in adsorption and removal of the analyte from tap water enriched with 0.5 mg L−1 of CDNB (Table 3). As shown in Table 3, the magnetic-MIP showed higher efficiency than the magnetic-NIP in the adsorption of CDNB, together with 0 100 200 300 400 0 2 4 6 Q e (m g g- 1 ) C e (mg L-1) (c) (b) (a) Fig. 11.Adsorption isotherms for (a) 1-chloro-2,4-dinitrobenzene, (b) 3,5-dichlorophenol, and (c) o-nitrophenol, obtained under the experimental conditions described in Section 2.7. complete desorption of the analyte after elution with methanol for 2 h. These results revealed that the magnetic-MIP synthesized in this work is a highly promisingmaterial for the recognition and selective re- moval of the CDNB molecule from water samples. 4. Conclusions This work describes a new magnetic-MIP selective to 1-chloro-2,4- dinitrobenzene (CDNB), a powerful allergenic compound that is widely used in various areas and can cause damage to health and the environ- ment. The preparedmaterial exhibited strongmagnetic responsiveness, good reproducibility, high adsorption capacity, and excellent selectivity. The magnetic properties of magnetite enabled the direct capture and easy separation of the magnetic-MIP from samples using an external magnetic field. The synthesized material showed good performance, with a high recovery value (near to 84%) and satisfactory extraction of CDNB from water samples. In addition, under appropriate conditions (and with the necessary ethical permission), it should be possible to use thismaterial in applications involving biological fluids such as urine. Acknowledgements The authors are grateful for the financial support provided by the Brazilian National Council for Scientific and Technological Development (CNPq), under the Science Without Borders program (processes Table 3 Recovery values obtained in adsorption and desorption experiments carried out to evalu- ate the efficiency of the magnetic materials. Material CCDBN = 0.5 mg L−1 Adsorption Desorption Magnetic-MIP 83.8 ± 0.8a 100 ± 1 Magnetic-NIP 66 ± 1 63.7 ± 0.8 a Relative deviations corresponding to triplicates. 372 R.J. Uzuriaga-Sánchez et al. / Materials Science and Engineering C 74 (2017) 365–373 4004759/2012-4 and 303979/2012-7), and InnovatePeru, under the Programa Nacional de Innovación para Competitividad y Productividad (Convenio No 513-INNOVATEPERU-ECIP-2015). 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Preparation of supermagnetic magnetite–OA nanoparticles 2.3. Encapsulation of magnetite-OA nanoparticles in a polymeric matrix: Influence of the functional monomer on MIP efficiency 2.4. Synthesis of magnetic–MIP selective to CDNB 2.5. Characterization of the magnetic-MIPs 2.5.1. X-ray diffraction (XRD) 2.5.2. Determination of specific surface area and porosity 2.5.3. Scanning electron microscopy (SEM) 2.5.4. Transmission electron microscopy (TEM) 2.6. Quantification of CDNB by HPLC 2.7. Binding and selective adsorption experiments 3. Results and discussion 3.1. Structural and morphological characterization of the magnetite 3.2. Influence of the functional monomer in the hydrophobic polymeric matrix of poly-(FM-co-EDGMA) on the magnetic-MIP efficiency 3.3. Morphological characterization of the magnetic-MIP for CDNB 3.4. Optimization of the adsorption capacity of the magnetic-MIP for CDNB 3.5. Selectivity studies 3.6. Analytical characteristics and application of the magnetic-MIP 4. Conclusions Acknowledgements Appendix A. Supplementary data References