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Computational simulation and preparation of fluorescent magnetic molecularly imprinted silica nanospheres for ciprofloxacin or norfloxacin sensing
Bo Gaoa,b, Xin-Ping Hea, Yang Jianga, Jia-Tong Weia, Hui Suoa,* , Chun Zhaoa a
State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and
Engineering, Jilin University, Changchun, 130012, P. R . China. b
Department of Resources and Environment , Jilin Agriculture University, Changchun,
130018, P. R . China. *Corresponding author. Tel: +86-431-85168241. Fax: +86-431-85112355. E-mail address: [email protected]
Received: 15-Sep-2014; Revised: 02-Oct-2014; Accepted: 03-Oct-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401014. This article is protected by copyright. All rights reserved.
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Abstract A magnetic molecularly imprinted fluorescent sensor for the sensitive and convenient determination of ciprofloxacin or norfloxacin in human urine was synthesized and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, Xray diffraction, ultraviolet/visible spectroscopy and fluorescence spectroscopy. Both cadmium telluride quantum dots and ferroferric oxide nanoparticles are introduced into the polymer for rapid separation and detection of the target molecules. The synthesized molecularly imprinted polymers were applied to detect ciprofloxacin or its structural analog norfloxacin in human urine with the detection limit 130 ng mL-1. A computational study was developed in order to evaluate the template-monomer geometry and interaction energy in the polymerization mixture to determine the reaction molar ratio of the template and monomer molecules.
Keywords: fluorescence; fluoroquinolones; magnetic separation ; molecular imprinting; molecular modeling;
1. Introduction Fluoroquinolones (FQs), such as the examples in Scheme S1, synthetic broad-spectrum chemotherapeutic bactericidal drugs, are widely used as human and veterinary medicines for treatment of pulmonary, urinary and digestive infections. However, they are also found to affect mammalian cell replication and associated with a great number of adverse drug reactions such as their latent allergic hypersensitivity reactions or toxic effects on the articular This article is protected by copyright. All rights reserved.
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cartilages causing arthralgia [1]. Therefore, monitoring FQs residues in biological samples and animal products is of great importance. Several methods for determining the residues of FQs have been developed in recent years, such as spectrophotometry [2], CE [3,4] and LC methods [5,6]. Most of these methods involve two steps including a preliminary extraction and a second clean-up step with LLE or SPE. As such, these methods will not only endure a complicated and time-consuming process but also use enormous amounts of organic solvents. Molecularly imprinted polymers (MIPs) have notable recognition properties for their templates. MIPs are prepared by the copolymerization of functional and cross-linking monomers in the presence of a template molecule [7,8]. After polymerization, removal of the template molecules leaves cavities that can selectively rebind template molecule on size, shape and functionality [9]. However, an MIP synthesized by conventional methods in bulk thermal- or photopolymerization is a monolithic polymer that should be ground and sieved, which results in irregularly shaped materials and the template and the binding sites are totally embedded in the polymer matrix, resulting in low mass transfer kinetics [10–12]. These problems can be overcome by the surface imprinting technique. Surface imprinted polymers have many advantages such as easier extraction of the original template, smaller particle size, larger surface area, higher binding capacity and faster mass transfer [13–16]. To improve the properties of MIPs, applying computational approach to molecular imprinting technique has already been attempted, as reported by many researchers such as Piletsky and et al., which makes it possible to select the monomer, solvent, cross-linker, and their proportion for a specific template molecule [17–23]. These approaches use different molecular modeling software to calculate binding energies and predict template–monomer This article is protected by copyright. All rights reserved.
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interaction positions in MIPs, which makes it easier to select the best functional monomer to be used and predict the best proportion of template-monomer in MIPs. Until now, most of these computational approaches are applied in the molecular imprinting modeling that is appropriate for the MIPs synthesized through conventional method, but the modeling of surface imprinting process remains unexplored. Herein, we set up the surface molecular imprinting model and apply quantum chemical computation to investigate the interactions between the template molecules and functional monomers. With the aid of theoretical calculations, we obtained a highly selective magnetic molecularly imprinted fluorescent sensor for ciprofloxacin (CFX) or norfloxacin (NFX). CdTe quantum dots (QDs), a fluorescent nanomaterial with size-dependent emission wavelengths, high luminescence efficiency, good photostability and tunable emission peaks, which makes them possible as sensing and recognizing elements [24–26], was used as a signal transducer in the synthesized MIPs. Meanwhile, encapsulating magnetic Fe3O4 nanoparticles into MIPs gives them magnetically susceptible characteristics and provide magnetic separation, which is easier and more convenient than the centrifugation and filtration step [27–29]. 2. Materials and methods 2.1 Reagents All the reagents in the experiments were of analytical grade and used as received without any further purification. Ciprofloxacin (CFX), ofloxacin (OFX), norfloxacin (NFX) and levofloxacin (LVFX) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Na2TeO3, 3-Mercaptopropionic This article is protected by copyright. All rights reserved.
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Acid (MPA) and 3-aminopropyltriethoxysilane (APTES) were procured from Aladdin Chemicals. CdCl2·2.5H2O, NaBH4, Hexadecyl trimethyl ammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), ethanol, oleic acid, (NH4)2Fe(SO4)2·6H2O, NaOH, ethyl acetate,
NH3·H2O (25%) were purchased from Beijing Chemical Factory. Ultrapure water with resistivity 18MΩcm-1 was used throughout. 2.2 Characterizations The X-ray diffraction (XRD) patterns were measured on a Ni-filtered X-ray diffractometer with CuKα radiation at 40 kV and 200 mA (Japan). FTIR spectra were measured by using a Bruker VERTEX-70 IR spectrometer. UV-visible spectra were recorded on a UV-2400 spectrophotometer (Shanghai, China). Fluorescence spectra were collected on a Shimadzu RF-5301 PC spectrophotometer equipped with a xenon lamp using right-angled geometry (Japan). The morphologies were performed on transmission electron microscope (TEM, JEOL JEM-2100) and a field-emission SEM (FE-SEM, JSM-6700F, JEOL, Japan). 2.3. Optimization of geometries and calculation of energies Density functional theory (DFT) was employed to optimize CFX molecule (Scheme S2) at the LC-WPBE/6-31G(d,p) level, the results of which and the experimental data [30] of CFX are presented in Table S1. It indicated that the LC-WPBE method was reliable in comparison with the crystal data. The optimized results proved the LC-WPBE/6-31G(d,p) was a reasonable method for geometry optimization. Thus, the LC-WPBE/6-31G(d,p) method was used for all the geometry optimizations in the rest of this study. To understand the template–monomer interactions at molecular level, the model of the template–monomer complexes were set up. Then all calculations had been carried out using This article is protected by copyright. All rights reserved.
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the Gaussian09 software with the Linux operating system. Electronic energies were calculated at the LC-WPBE level with the 6-31G(d,p) basis set. For the selected functional monomer, the most stable template–monomer complexes were searched and their interaction energy, ΔE, was calculated as follows: ΔE=E(template–monomer) – nE(monomer) – nE(template) (1) The Millikan charge was used to perform the charge distribution. To explore the strength of hydrogen bonding interactions, we calculated the hydrogen bond length of a stable complex formed from different numbers of CFX molecules and the functional monomers. 2.4 Synthesis of MIP and NIP nanoparticles Firstly, Fe3O4 nanoparticles capped with oleic acid and CdTe QDs were synthesized according to the literature [31] and [32, 33], respectively with some modification. CdTe QDs with maximum emission wavelength at 580nm was used in the following experiments. The CdTe QDs were precipitated with ethanol, separated by centrifugation and dispersed in 30 mL water for use. For XRD measurement, QDs powder was obtained by drying the precipitation in vacuum oven. Fe3O4 nanoparticles capped with oleic acid were transferred into water soluble Fe3O4 as follows: one milliliter of chloroform of Fe3O4 (10 mg) was added to a 10 mL CTAB (0.4g) aqueous solution with ultrasonication until a homogeneous Fe3O4 aqueous solution was obtained. Secondly, the MIP nanoparticles were synthesized as follows: 0.8 mL 25% NH3·H2O, 2.5 mL ethyl acetate and 5 mL Fe3O4 aqueous solution were added into 30 mL CdTe QDs aqueous solution and mixed under mechanical stirring. Then, a mixture with 18 μL APTES and 13 mg CFX (nAPTES/nCFX =2:1) in 8 mL aqueous was added, stirring for 15 min. This article is protected by copyright. All rights reserved.
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Subsequently, 100 μL TEOS was added dropwise. The resultant mixture was stirred for 3 h under room temperature. The template was removed by washing with absolute ethanol three times. For comparison, nonimprinted polymers (NIPs) were synthesized similarly but without adding CFX. Finally, the MIPs and NIPs were isolated by magnetic separation, washed with ethanol and dried in a vacuum oven. 2.5 Fluorescence measurements All fluorescence detections were performed under the same conditions: the slit widths of excitation and emission were 5 and 10 nm, respectively. The excitation wavelength was set at 415 nm with a recording emission range of 450–700 nm. To a 10 mL calibrated test tube, MIPs or NIPs powder (6.25 mg), 1 mL of Tris-HCl (pH=7.4) buffer solution and a desired concentration of CFX standard solution or aqueous sample solution were sequentially added. The mixture was then diluted to volume with ultrapure water and incubated at room temperature for 20 min and then ultrasonicated for 5 min before scanned with an RF-5301PC spectrophotometer. 3. Results and discussion 3.1 Optimization of geometries and calculation of energies A silane coupling agent was needed to be used as functional monomer. APTES is a good candidate due to its ability of forming strong hydrogen bonds in –NH2 group with other template molecules. TEOS was chosen as cross-linker which will hydrolyze into SiO2 during reaction process. At the same time APTES will also hydrolyze to form three –OH terminal groups. Then, a condensation reaction will take place among the –OH groups of APTES monomers as well as the –OH groups at the surface of SiO2. Thus, a large number of This article is protected by copyright. All rights reserved.
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functional monomers with the terminal –NH2 bonds grafted on the surface of SiO2 (Fig. S1) and will function with the template molecules through hydrogen bonds. Because there are so many functional monomers grafted on the surface of SiO2, we can only select a fraction (six silanized APTES molecules graft on the surface of SiO2) to use for theoretical calculations, and optimized its conformation, as shown in Fig. S2a. The optimized conformation of CFX is shown in Fig. S2b. The Millikan charges of the possible proton acceptors and proton donors in CFX and the selected fraction of silanized APTES are signed out. We can see that the proton acceptors are F, O1 and O2 in CFX, and the proton donors are H1, H2, H3 and H4 in silanized APTES. On the basis of Millikan charges analyses of the proton donors and acceptors in silanized APTES and CFX, we simulated different ratios (6:1, 6:2 and 6:3) of APTES monomer and CFX template. Then the optimized template–monomer complex possessed the lowest total energy (for each ratio) and the maximum number of hydrogen bond was selected. The optimized complexes are presented in Fig. 1. To explore the strength of hydrogen bonding interactions, the possible conformations and hydrogen bonds of the stable complexes formed by silanized APTES and different numbers of CFX molecules were presented. We can see that hydrogen bonds marked with dotted line formed between the proton acceptors in CFX and the proton donors in silanized APTES. The numbers of hydrogen bonds increased along with the number of CFX template molecules interacting with the silanized APTES functional monomers, as shown in Fig. 1b and Fig. 1c (The color picture of Fig.1 is presented in supplementary information for clarity). DFT was employed to calculate the binding energies between APTES and CFX with This article is protected by copyright. All rights reserved.
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different molar ratios (6:1, 6:2, 6:3) at the LC-WPBE/6-31G(d,p) level, and the results are listed in Table 1. In general, the systematic energy decreases when two molecules interact with each other, which means that the process of the formation of complexes is a process of energy reducing. We can see from Table 1 that ΔE(complex
c)
(Fig.1c)>ΔE(complex
b)
(Fig.1b)>ΔE(complex a) (Fig.1a), which indicates that when the mole ratio of APTES and CFX was 6:3 (namely 2:1), the template–monomer complex possessed the lowest total energy and the maximum number of hydrogen bonds. Therefore, the suitable reaction mole ratio of APTES monomer and CFX template in the experiment was determined to be 2:1. 3.2 Preparation of MIPs and NIPs nanoparticles To confirm the reaction mole ratio of APTES and CFX, UV spectroscopy was used to explore prepolymerization aqueous solution for different mole ratio (6:1, 3:1, 2:1) of APTES and CFX, and the result was shown in Fig. S3. The UV absorption curve of CFX (curve 1) shows a strong absorption peak at 271 nm, which decreases when APTES was added in, indicating the molecular interaction between APTES monomers and CFX template molecules. When the mole ratio of the two species was 2:1, the absorption peak at 271 had the maximum decrease, suggesting more strengthened interaction between APTES and CFX. Thus, the reaction mole ratio of APTES and CFX was set as 2:1, which is in accordance with the above theoretical calculations. In this work, the water-soluble CdTe QDs were modified with MPA, which not only improved stability and dispersion of QDs but also served as specific monomer, providing – SH for the QDs to form the desired surface binding groups with MIPs. We introduced CdTe QDs and Fe3O4 nanoparticles into the MIPs, which could be separated with an external This article is protected by copyright. All rights reserved.
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magnetic field and monitor the template molecules by the changes of fluorescence intensity of CdTe QDs. The imprinting process is illustrated in Fig. 2. First, oleic acid-capped Fe3O4 nanoparticles were transferred into aqueous phase with the aid of CTAB. Afterward, water soluble Fe3O4 nanoparticles and CdTe QDs were embedded into the imprinted silica matrix with the sol–gel reaction of TEOS. The template molecules CFX and functional monomer APTES were preassembled by the ion pair interaction and fixed up into silica matrix under a sol–gel process with the assistance of the cross-linking agent TEOS. The silica nanospheres were simply fabricated by means of the hydrolysis and condensation reaction of APTES and TEOS in the presence of aqueous ammonia solution as the catalyst [34]. CFX templates were assembled and immobilized into the matrix of silica by silanization reaction between APTES and TEOS. After the templates were extracted from the silica matrix by using solvent to decompose the hydrogen bond, the CFX-imprinted sites with the covalently anchored amino groups at the cavity were created in the silica matrix. Finally, the MIPs with cavities complementary in shape and functionality to CFX were obtained after CFX was removed. 3.3 Characterization of MIPs nanoparticles Fig. S4 shows the XRD patterns of CdTe QDs, oleic acid capped Fe3O4 nanoparticles, and MIPs embedded CdTe QDs. Curve (a) is the XRD pattern of CdTe QDs, all diffraction peaks in which can be indexed to a cubic zinc blende structure. Curve (b) shows the XRD pattern of oleic acid capped Fe3O4 nanoparticles, which reveals a face centered cubic structure. The XRD pattern of MIPs is shown in curve (c), in which we could found characteristic peaks of CdTe QDs and Fe3O4 nanoparticles, demonstrating the presence of CdTe QDs and Fe3O4 nanoparticles in MIPs. The above conclusion can be further confirmed by the experiment This article is protected by copyright. All rights reserved.
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with an external magnetic field, as shown in inset in Fig. S4(c). The MIPs with an orange color (the color of CdTe QDs) moved to the magnet’s side within several seconds. Fig. 3 displays the HR-TEM images of CdTe QDs, TEM images of oleic acid capped Fe3O4 nanoparticles as well as the SEM and TEM images of MIPs nanoparticles. From Fig. 3a, CdTe QDs are well crystallized and nearly monodispersed with ellipsoidal shape in a diameter about 5 nm. Fig. 3b shows the TEM image of oleic acid capped Fe3O4 nanoparticles, which are well dispersive with a diameter range of 5–10 nm. The SEM image of MIPs nanoparticles displayed in Fig. 3c shows a spherical morphology with diameters of 50–100 nm and the particles aggregate a little. Fig. 3d shows the TEM image of MIPs nanoparticles, in which we can see Fe3O4 nanoparticles embedded in the MIPs particles and silica matrix with light shadow part. The FTIR spectra of MIPs and NIPs nanoparticles are compared in Fig. S5. The FTIR spectra of these two polymers showed major bands in similar locations because the compositions of MIPs and NIPs were similar. The broad and strong peak around 1065 cm-1 suggests Si–O–Si asymmetric stretching, and the bands around 795 cm-1 are attributed to Si– O vibrations. The peak located at 580 cm-1 is the characteristic band of Fe3O4, indicating the existence of Fe3O4 in these two polymers. The band around 3410 cm-1 is attributed to N–H, indicating the existence of amino groups in these two polymers. 3.4 Fluorescence properties of MIPs nanoparticles We used UV-vis and fluorescence spectroscopy to investigate the optical properties of CdTe QDs and MIPs. From Fig. S6a, we can see that the characteristic absorption peak of MIPs remains the same with CdTe QDs, which suggests CdTe QDs was successful attached This article is protected by copyright. All rights reserved.
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into MIPs. However, the peak of MIPs is less pronounced because of the broad and strong absorption of Fe3O4 [35–37]. From Fig. S6b, the prepared MIPs showed the same fluorescence emission peak at 580nm with CdTe QDs, which demonstrates the Fe3O4 encapsulated in the MIPs has little interference to the property of CdTe QDs. The RSD of ~ 2% was obtained by 12 further repeated detections of the fluorescence intensity in the 625 μg mL1
MIPs aqueous solution for every 5 min. The results shown in Fig. S6c indicate the stable
emission properties of the obtained MIPs. To study the kinetic binding behavior of MIPs for CFX, CFX (500 ng mL-1) was added to MIPs (625 μg mL-1) in water and the fluorescence spectroscopy was measured at 10 min intervals. The fluorescence intensity rapidly decreased up to 20 min and thereafter decreased slowly (Fig. S7). Therefore, the subsequent fluorescence quenching experiment was performed after the MIPs and CFX were incubated in the solution for 20 min. MIPs were used as a sensing material to detect CFX based on the fluorescence quenching between CFX and the CdTe QDs. We can see from Fig. 4, as the amount (50, 100, 150, 200, 300, 400, 500 and 600 ng mL-1) of CFX added into MIPs water solution, the fluorescence intensity decreases observably. The mechanism of the decrease in fluorescence intensity may involve charge transfer from CdTe QDs to CFX. Such a charge-transfer mechanism has been proposed by Tu et al [38]. The relationship between the fluorescence intensity and the concentration of quenching CFX can be described by the Stern–Volmer equation: (F0/F) =1+Ksv[A] (2) where F0 and F are the fluorescence intensities in the absence and presence of analyte, respectively, [A] is the concentration of analyte, and Ksv is the quenching constant. The This article is protected by copyright. All rights reserved.
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dependence of F0/F as a function of [A] is shown inset in Fig. 4. The Ksv of MIPs is found to be 4.8×103 L mol-1. The LOD, calculated following the 3σ IUPAC criteria, is 130ng mL-1. To demonstrate the imprinting effect, as a control experiment, the fluorescence response of NIPs to the template molecule is investigated. As shown in inset of Fig. 4, the Ksv of NIPs is 1.8×103 L mol-1. The Ksv of the MIPs or NIPs was important data to evaluate the selectivity and sensitivity of the materials we obtained. According to the above results, the Ksv for MIPs is about threefold of that for NIPs, demonstrating the MIPs have a better selectivity and sensitivity than the NIPs. We studied the fluorescence response of MIPs toward several other FQs to examine the selectivity of MIPs. As shown in Fig. S8, CFX, NFX, OFX and LVXF are selected to assess the selectivity of MIPs. We can see that, both CFX and NFX show strong fluorescence quenching of MIPs and exhibit comparative Ksv values. The quenching efficiency for MIPs is about threefold of that for NIPs. However, OFX and LVXF display little fluorescence response to both MIPs and NIPs. For the above results, we propose the following possibilities: MIPs with proper cavities can provide a better space to rebind the target molecules; after rebinding, the target molecules could cause the charge-transfer efficiently and result in the increase of efficiency of fluorescence quenching. The fluorescence titration of MIPs with common anion and ion was conducted to examine the interference of ions. As shown in Table S2, the interference of alkali, alkali earth ions and anions, such as Na+, K+, Ca2+, Mg2+, NO3-, CO32-, and so forth have basically no effect on fluorescence intensity of MIPs. 3.5 Application to human sample analysis Human urine samples were collected from healthy volunteers and stored at –20°C in a This article is protected by copyright. All rights reserved.
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freezer until use. The urine sample was filtered through 0.45 μm Supor filters and stored in precleaned glass bottles. Because no CFX and NFX in the collected urine samples were detectable by the proposed method, a recovery study was carried out on the samples spiked with 500 ng mL-1 CFX and 400 ng mL-1 NFX separately to evaluate the developed method. The results are shown in Table S3. The apparent recoveries were 97 and 98% for CFX and NFX, respectively. This result demonstrates that the MIPs can measure the CFX or NFX concentrations, respectively, in human urine samples. 4. Conclusion The computational method was used to investigate the interactions between template molecules and functional monomers during surface imprinting process. With the theoretical computational results, the ratio of template and monomer was determined, and then a magnetic molecularly imprinted silica nanoparticle embedded CdTe QDs was prepared as a fluorescent sensor. The synthesized MIPs exhibit good molecular recognition properties in terms of both sensitivity and selectivity for NFX or CFX. Because these two antibiotics are of the same kind, usually they are not used at the same time for human. Thus, the synthesized fluorescent MIPs are suitable for the selective detection of NFX and CFX, respectively, in human urine samples. Furthermore, molecular recognition of magnetic fluorescent sensor as well the corresponding computational modeling is in progress in our laboratory, which should spark a broad spectrum of interest due to its great versatility for future applications.
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Acknowledgment This work was supported by State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Natural Science Foundation of P. R. China (Item No. 61374218).
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Fig. 1 Models of the complexes formed from different numbers of CFX molecules and silanized APTES. (Si gray/blue; N blue; O red; C gray; H white; F green)
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Fig. 2 Schematic procedure of imprinting CFX in a silica matrix embedded with Fe3O4 nanoparticles and CdTe QDs.
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Fig. 3 HR-TEM images of CdTe QDs (a), TEM images of oleic acid capped Fe3O4 nanoparticles (b) and SEM image (c) and TEM image (d) of MIPs.
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Fig. 4 Fluorescence spectroscopy of the MIPs aqueous solution with the increasing concentrations of CFX adding in. Inset: Stern–Volmer-type description of the data showing a linear fit throughout the CFX concentration range.
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Table 1. Binding Energies (in KJ mol-1) of silanized APTES model with different numbers of CFX molecules.
Species CFX
silanized APTES Complex (a) Complex (b) Complex (c)
E –3013224.0990 –20237143.3304 –23250405.7616 –26263681.8455 –29276963.7054
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ΔE
– – –38.3322 –90.3172 –148.0782
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Computational simulation and preparation of fluorescent magnetic molecularly imprinted silica nanospheres for ciprofloxacin or norfloxacin sensing
Bo Gaoa,b, Xin-Ping Hea, Yang Jianga, Jia-Tong Weia, Hui Suoa,* , Chun Zhaoa a
State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and
Engineering, Jilin University, Changchun, 130012, P. R . China. b
Department of Resources and Environment , Jilin Agriculture University, Changchun,
130018, P. R . China. *Corresponding author. Tel: +86-431-85168241. Fax: +86-431-85112355. E-mail address: [email protected]
Received: 15-Sep-2014; Revised: 02-Oct-2014; Accepted: 03-Oct-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401014. This article is protected by copyright. All rights reserved.
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Abstract A magnetic molecularly imprinted fluorescent sensor for the sensitive and convenient determination of ciprofloxacin or norfloxacin in human urine was synthesized and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, Xray diffraction, ultraviolet/visible spectroscopy and fluorescence spectroscopy. Both cadmium telluride quantum dots and ferroferric oxide nanoparticles are introduced into the polymer for rapid separation and detection of the target molecules. The synthesized molecularly imprinted polymers were applied to detect ciprofloxacin or its structural analog norfloxacin in human urine with the detection limit 130 ng mL-1. A computational study was developed in order to evaluate the template-monomer geometry and interaction energy in the polymerization mixture to determine the reaction molar ratio of the template and monomer molecules.
Keywords: fluorescence; fluoroquinolones; magnetic separation ; molecular imprinting; molecular modeling;
1. Introduction Fluoroquinolones (FQs), such as the examples in Scheme S1, synthetic broad-spectrum chemotherapeutic bactericidal drugs, are widely used as human and veterinary medicines for treatment of pulmonary, urinary and digestive infections. However, they are also found to affect mammalian cell replication and associated with a great number of adverse drug reactions such as their latent allergic hypersensitivity reactions or toxic effects on the articular This article is protected by copyright. All rights reserved.
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cartilages causing arthralgia [1]. Therefore, monitoring FQs residues in biological samples and animal products is of great importance. Several methods for determining the residues of FQs have been developed in recent years, such as spectrophotometry [2], CE [3,4] and LC methods [5,6]. Most of these methods involve two steps including a preliminary extraction and a second clean-up step with LLE or SPE. As such, these methods will not only endure a complicated and time-consuming process but also use enormous amounts of organic solvents. Molecularly imprinted polymers (MIPs) have notable recognition properties for their templates. MIPs are prepared by the copolymerization of functional and cross-linking monomers in the presence of a template molecule [7,8]. After polymerization, removal of the template molecules leaves cavities that can selectively rebind template molecule on size, shape and functionality [9]. However, an MIP synthesized by conventional methods in bulk thermal- or photopolymerization is a monolithic polymer that should be ground and sieved, which results in irregularly shaped materials and the template and the binding sites are totally embedded in the polymer matrix, resulting in low mass transfer kinetics [10–12]. These problems can be overcome by the surface imprinting technique. Surface imprinted polymers have many advantages such as easier extraction of the original template, smaller particle size, larger surface area, higher binding capacity and faster mass transfer [13–16]. To improve the properties of MIPs, applying computational approach to molecular imprinting technique has already been attempted, as reported by many researchers such as Piletsky and et al., which makes it possible to select the monomer, solvent, cross-linker, and their proportion for a specific template molecule [17–23]. These approaches use different molecular modeling software to calculate binding energies and predict template–monomer This article is protected by copyright. All rights reserved.
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interaction positions in MIPs, which makes it easier to select the best functional monomer to be used and predict the best proportion of template-monomer in MIPs. Until now, most of these computational approaches are applied in the molecular imprinting modeling that is appropriate for the MIPs synthesized through conventional method, but the modeling of surface imprinting process remains unexplored. Herein, we set up the surface molecular imprinting model and apply quantum chemical computation to investigate the interactions between the template molecules and functional monomers. With the aid of theoretical calculations, we obtained a highly selective magnetic molecularly imprinted fluorescent sensor for ciprofloxacin (CFX) or norfloxacin (NFX). CdTe quantum dots (QDs), a fluorescent nanomaterial with size-dependent emission wavelengths, high luminescence efficiency, good photostability and tunable emission peaks, which makes them possible as sensing and recognizing elements [24–26], was used as a signal transducer in the synthesized MIPs. Meanwhile, encapsulating magnetic Fe3O4 nanoparticles into MIPs gives them magnetically susceptible characteristics and provide magnetic separation, which is easier and more convenient than the centrifugation and filtration step [27–29]. 2. Materials and methods 2.1 Reagents All the reagents in the experiments were of analytical grade and used as received without any further purification. Ciprofloxacin (CFX), ofloxacin (OFX), norfloxacin (NFX) and levofloxacin (LVFX) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Na2TeO3, 3-Mercaptopropionic This article is protected by copyright. All rights reserved.
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Acid (MPA) and 3-aminopropyltriethoxysilane (APTES) were procured from Aladdin Chemicals. CdCl2·2.5H2O, NaBH4, Hexadecyl trimethyl ammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), ethanol, oleic acid, (NH4)2Fe(SO4)2·6H2O, NaOH, ethyl acetate,
NH3·H2O (25%) were purchased from Beijing Chemical Factory. Ultrapure water with resistivity 18MΩcm-1 was used throughout. 2.2 Characterizations The X-ray diffraction (XRD) patterns were measured on a Ni-filtered X-ray diffractometer with CuKα radiation at 40 kV and 200 mA (Japan). FTIR spectra were measured by using a Bruker VERTEX-70 IR spectrometer. UV-visible spectra were recorded on a UV-2400 spectrophotometer (Shanghai, China). Fluorescence spectra were collected on a Shimadzu RF-5301 PC spectrophotometer equipped with a xenon lamp using right-angled geometry (Japan). The morphologies were performed on transmission electron microscope (TEM, JEOL JEM-2100) and a field-emission SEM (FE-SEM, JSM-6700F, JEOL, Japan). 2.3. Optimization of geometries and calculation of energies Density functional theory (DFT) was employed to optimize CFX molecule (Scheme S2) at the LC-WPBE/6-31G(d,p) level, the results of which and the experimental data [30] of CFX are presented in Table S1. It indicated that the LC-WPBE method was reliable in comparison with the crystal data. The optimized results proved the LC-WPBE/6-31G(d,p) was a reasonable method for geometry optimization. Thus, the LC-WPBE/6-31G(d,p) method was used for all the geometry optimizations in the rest of this study. To understand the template–monomer interactions at molecular level, the model of the template–monomer complexes were set up. Then all calculations had been carried out using This article is protected by copyright. All rights reserved.
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the Gaussian09 software with the Linux operating system. Electronic energies were calculated at the LC-WPBE level with the 6-31G(d,p) basis set. For the selected functional monomer, the most stable template–monomer complexes were searched and their interaction energy, ΔE, was calculated as follows: ΔE=E(template–monomer) – nE(monomer) – nE(template) (1) The Millikan charge was used to perform the charge distribution. To explore the strength of hydrogen bonding interactions, we calculated the hydrogen bond length of a stable complex formed from different numbers of CFX molecules and the functional monomers. 2.4 Synthesis of MIP and NIP nanoparticles Firstly, Fe3O4 nanoparticles capped with oleic acid and CdTe QDs were synthesized according to the literature [31] and [32, 33], respectively with some modification. CdTe QDs with maximum emission wavelength at 580nm was used in the following experiments. The CdTe QDs were precipitated with ethanol, separated by centrifugation and dispersed in 30 mL water for use. For XRD measurement, QDs powder was obtained by drying the precipitation in vacuum oven. Fe3O4 nanoparticles capped with oleic acid were transferred into water soluble Fe3O4 as follows: one milliliter of chloroform of Fe3O4 (10 mg) was added to a 10 mL CTAB (0.4g) aqueous solution with ultrasonication until a homogeneous Fe3O4 aqueous solution was obtained. Secondly, the MIP nanoparticles were synthesized as follows: 0.8 mL 25% NH3·H2O, 2.5 mL ethyl acetate and 5 mL Fe3O4 aqueous solution were added into 30 mL CdTe QDs aqueous solution and mixed under mechanical stirring. Then, a mixture with 18 μL APTES and 13 mg CFX (nAPTES/nCFX =2:1) in 8 mL aqueous was added, stirring for 15 min. This article is protected by copyright. All rights reserved.
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Subsequently, 100 μL TEOS was added dropwise. The resultant mixture was stirred for 3 h under room temperature. The template was removed by washing with absolute ethanol three times. For comparison, nonimprinted polymers (NIPs) were synthesized similarly but without adding CFX. Finally, the MIPs and NIPs were isolated by magnetic separation, washed with ethanol and dried in a vacuum oven. 2.5 Fluorescence measurements All fluorescence detections were performed under the same conditions: the slit widths of excitation and emission were 5 and 10 nm, respectively. The excitation wavelength was set at 415 nm with a recording emission range of 450–700 nm. To a 10 mL calibrated test tube, MIPs or NIPs powder (6.25 mg), 1 mL of Tris-HCl (pH=7.4) buffer solution and a desired concentration of CFX standard solution or aqueous sample solution were sequentially added. The mixture was then diluted to volume with ultrapure water and incubated at room temperature for 20 min and then ultrasonicated for 5 min before scanned with an RF-5301PC spectrophotometer. 3. Results and discussion 3.1 Optimization of geometries and calculation of energies A silane coupling agent was needed to be used as functional monomer. APTES is a good candidate due to its ability of forming strong hydrogen bonds in –NH2 group with other template molecules. TEOS was chosen as cross-linker which will hydrolyze into SiO2 during reaction process. At the same time APTES will also hydrolyze to form three –OH terminal groups. Then, a condensation reaction will take place among the –OH groups of APTES monomers as well as the –OH groups at the surface of SiO2. Thus, a large number of This article is protected by copyright. All rights reserved.
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functional monomers with the terminal –NH2 bonds grafted on the surface of SiO2 (Fig. S1) and will function with the template molecules through hydrogen bonds. Because there are so many functional monomers grafted on the surface of SiO2, we can only select a fraction (six silanized APTES molecules graft on the surface of SiO2) to use for theoretical calculations, and optimized its conformation, as shown in Fig. S2a. The optimized conformation of CFX is shown in Fig. S2b. The Millikan charges of the possible proton acceptors and proton donors in CFX and the selected fraction of silanized APTES are signed out. We can see that the proton acceptors are F, O1 and O2 in CFX, and the proton donors are H1, H2, H3 and H4 in silanized APTES. On the basis of Millikan charges analyses of the proton donors and acceptors in silanized APTES and CFX, we simulated different ratios (6:1, 6:2 and 6:3) of APTES monomer and CFX template. Then the optimized template–monomer complex possessed the lowest total energy (for each ratio) and the maximum number of hydrogen bond was selected. The optimized complexes are presented in Fig. 1. To explore the strength of hydrogen bonding interactions, the possible conformations and hydrogen bonds of the stable complexes formed by silanized APTES and different numbers of CFX molecules were presented. We can see that hydrogen bonds marked with dotted line formed between the proton acceptors in CFX and the proton donors in silanized APTES. The numbers of hydrogen bonds increased along with the number of CFX template molecules interacting with the silanized APTES functional monomers, as shown in Fig. 1b and Fig. 1c (The color picture of Fig.1 is presented in supplementary information for clarity). DFT was employed to calculate the binding energies between APTES and CFX with This article is protected by copyright. All rights reserved.
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different molar ratios (6:1, 6:2, 6:3) at the LC-WPBE/6-31G(d,p) level, and the results are listed in Table 1. In general, the systematic energy decreases when two molecules interact with each other, which means that the process of the formation of complexes is a process of energy reducing. We can see from Table 1 that ΔE(complex
c)
(Fig.1c)>ΔE(complex
b)
(Fig.1b)>ΔE(complex a) (Fig.1a), which indicates that when the mole ratio of APTES and CFX was 6:3 (namely 2:1), the template–monomer complex possessed the lowest total energy and the maximum number of hydrogen bonds. Therefore, the suitable reaction mole ratio of APTES monomer and CFX template in the experiment was determined to be 2:1. 3.2 Preparation of MIPs and NIPs nanoparticles To confirm the reaction mole ratio of APTES and CFX, UV spectroscopy was used to explore prepolymerization aqueous solution for different mole ratio (6:1, 3:1, 2:1) of APTES and CFX, and the result was shown in Fig. S3. The UV absorption curve of CFX (curve 1) shows a strong absorption peak at 271 nm, which decreases when APTES was added in, indicating the molecular interaction between APTES monomers and CFX template molecules. When the mole ratio of the two species was 2:1, the absorption peak at 271 had the maximum decrease, suggesting more strengthened interaction between APTES and CFX. Thus, the reaction mole ratio of APTES and CFX was set as 2:1, which is in accordance with the above theoretical calculations. In this work, the water-soluble CdTe QDs were modified with MPA, which not only improved stability and dispersion of QDs but also served as specific monomer, providing – SH for the QDs to form the desired surface binding groups with MIPs. We introduced CdTe QDs and Fe3O4 nanoparticles into the MIPs, which could be separated with an external This article is protected by copyright. All rights reserved.
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magnetic field and monitor the template molecules by the changes of fluorescence intensity of CdTe QDs. The imprinting process is illustrated in Fig. 2. First, oleic acid-capped Fe3O4 nanoparticles were transferred into aqueous phase with the aid of CTAB. Afterward, water soluble Fe3O4 nanoparticles and CdTe QDs were embedded into the imprinted silica matrix with the sol–gel reaction of TEOS. The template molecules CFX and functional monomer APTES were preassembled by the ion pair interaction and fixed up into silica matrix under a sol–gel process with the assistance of the cross-linking agent TEOS. The silica nanospheres were simply fabricated by means of the hydrolysis and condensation reaction of APTES and TEOS in the presence of aqueous ammonia solution as the catalyst [34]. CFX templates were assembled and immobilized into the matrix of silica by silanization reaction between APTES and TEOS. After the templates were extracted from the silica matrix by using solvent to decompose the hydrogen bond, the CFX-imprinted sites with the covalently anchored amino groups at the cavity were created in the silica matrix. Finally, the MIPs with cavities complementary in shape and functionality to CFX were obtained after CFX was removed. 3.3 Characterization of MIPs nanoparticles Fig. S4 shows the XRD patterns of CdTe QDs, oleic acid capped Fe3O4 nanoparticles, and MIPs embedded CdTe QDs. Curve (a) is the XRD pattern of CdTe QDs, all diffraction peaks in which can be indexed to a cubic zinc blende structure. Curve (b) shows the XRD pattern of oleic acid capped Fe3O4 nanoparticles, which reveals a face centered cubic structure. The XRD pattern of MIPs is shown in curve (c), in which we could found characteristic peaks of CdTe QDs and Fe3O4 nanoparticles, demonstrating the presence of CdTe QDs and Fe3O4 nanoparticles in MIPs. The above conclusion can be further confirmed by the experiment This article is protected by copyright. All rights reserved.
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with an external magnetic field, as shown in inset in Fig. S4(c). The MIPs with an orange color (the color of CdTe QDs) moved to the magnet’s side within several seconds. Fig. 3 displays the HR-TEM images of CdTe QDs, TEM images of oleic acid capped Fe3O4 nanoparticles as well as the SEM and TEM images of MIPs nanoparticles. From Fig. 3a, CdTe QDs are well crystallized and nearly monodispersed with ellipsoidal shape in a diameter about 5 nm. Fig. 3b shows the TEM image of oleic acid capped Fe3O4 nanoparticles, which are well dispersive with a diameter range of 5–10 nm. The SEM image of MIPs nanoparticles displayed in Fig. 3c shows a spherical morphology with diameters of 50–100 nm and the particles aggregate a little. Fig. 3d shows the TEM image of MIPs nanoparticles, in which we can see Fe3O4 nanoparticles embedded in the MIPs particles and silica matrix with light shadow part. The FTIR spectra of MIPs and NIPs nanoparticles are compared in Fig. S5. The FTIR spectra of these two polymers showed major bands in similar locations because the compositions of MIPs and NIPs were similar. The broad and strong peak around 1065 cm-1 suggests Si–O–Si asymmetric stretching, and the bands around 795 cm-1 are attributed to Si– O vibrations. The peak located at 580 cm-1 is the characteristic band of Fe3O4, indicating the existence of Fe3O4 in these two polymers. The band around 3410 cm-1 is attributed to N–H, indicating the existence of amino groups in these two polymers. 3.4 Fluorescence properties of MIPs nanoparticles We used UV-vis and fluorescence spectroscopy to investigate the optical properties of CdTe QDs and MIPs. From Fig. S6a, we can see that the characteristic absorption peak of MIPs remains the same with CdTe QDs, which suggests CdTe QDs was successful attached This article is protected by copyright. All rights reserved.
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into MIPs. However, the peak of MIPs is less pronounced because of the broad and strong absorption of Fe3O4 [35–37]. From Fig. S6b, the prepared MIPs showed the same fluorescence emission peak at 580nm with CdTe QDs, which demonstrates the Fe3O4 encapsulated in the MIPs has little interference to the property of CdTe QDs. The RSD of ~ 2% was obtained by 12 further repeated detections of the fluorescence intensity in the 625 μg mL1
MIPs aqueous solution for every 5 min. The results shown in Fig. S6c indicate the stable
emission properties of the obtained MIPs. To study the kinetic binding behavior of MIPs for CFX, CFX (500 ng mL-1) was added to MIPs (625 μg mL-1) in water and the fluorescence spectroscopy was measured at 10 min intervals. The fluorescence intensity rapidly decreased up to 20 min and thereafter decreased slowly (Fig. S7). Therefore, the subsequent fluorescence quenching experiment was performed after the MIPs and CFX were incubated in the solution for 20 min. MIPs were used as a sensing material to detect CFX based on the fluorescence quenching between CFX and the CdTe QDs. We can see from Fig. 4, as the amount (50, 100, 150, 200, 300, 400, 500 and 600 ng mL-1) of CFX added into MIPs water solution, the fluorescence intensity decreases observably. The mechanism of the decrease in fluorescence intensity may involve charge transfer from CdTe QDs to CFX. Such a charge-transfer mechanism has been proposed by Tu et al [38]. The relationship between the fluorescence intensity and the concentration of quenching CFX can be described by the Stern–Volmer equation: (F0/F) =1+Ksv[A] (2) where F0 and F are the fluorescence intensities in the absence and presence of analyte, respectively, [A] is the concentration of analyte, and Ksv is the quenching constant. The This article is protected by copyright. All rights reserved.
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dependence of F0/F as a function of [A] is shown inset in Fig. 4. The Ksv of MIPs is found to be 4.8×103 L mol-1. The LOD, calculated following the 3σ IUPAC criteria, is 130ng mL-1. To demonstrate the imprinting effect, as a control experiment, the fluorescence response of NIPs to the template molecule is investigated. As shown in inset of Fig. 4, the Ksv of NIPs is 1.8×103 L mol-1. The Ksv of the MIPs or NIPs was important data to evaluate the selectivity and sensitivity of the materials we obtained. According to the above results, the Ksv for MIPs is about threefold of that for NIPs, demonstrating the MIPs have a better selectivity and sensitivity than the NIPs. We studied the fluorescence response of MIPs toward several other FQs to examine the selectivity of MIPs. As shown in Fig. S8, CFX, NFX, OFX and LVXF are selected to assess the selectivity of MIPs. We can see that, both CFX and NFX show strong fluorescence quenching of MIPs and exhibit comparative Ksv values. The quenching efficiency for MIPs is about threefold of that for NIPs. However, OFX and LVXF display little fluorescence response to both MIPs and NIPs. For the above results, we propose the following possibilities: MIPs with proper cavities can provide a better space to rebind the target molecules; after rebinding, the target molecules could cause the charge-transfer efficiently and result in the increase of efficiency of fluorescence quenching. The fluorescence titration of MIPs with common anion and ion was conducted to examine the interference of ions. As shown in Table S2, the interference of alkali, alkali earth ions and anions, such as Na+, K+, Ca2+, Mg2+, NO3-, CO32-, and so forth have basically no effect on fluorescence intensity of MIPs. 3.5 Application to human sample analysis Human urine samples were collected from healthy volunteers and stored at –20°C in a This article is protected by copyright. All rights reserved.
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freezer until use. The urine sample was filtered through 0.45 μm Supor filters and stored in precleaned glass bottles. Because no CFX and NFX in the collected urine samples were detectable by the proposed method, a recovery study was carried out on the samples spiked with 500 ng mL-1 CFX and 400 ng mL-1 NFX separately to evaluate the developed method. The results are shown in Table S3. The apparent recoveries were 97 and 98% for CFX and NFX, respectively. This result demonstrates that the MIPs can measure the CFX or NFX concentrations, respectively, in human urine samples. 4. Conclusion The computational method was used to investigate the interactions between template molecules and functional monomers during surface imprinting process. With the theoretical computational results, the ratio of template and monomer was determined, and then a magnetic molecularly imprinted silica nanoparticle embedded CdTe QDs was prepared as a fluorescent sensor. The synthesized MIPs exhibit good molecular recognition properties in terms of both sensitivity and selectivity for NFX or CFX. Because these two antibiotics are of the same kind, usually they are not used at the same time for human. Thus, the synthesized fluorescent MIPs are suitable for the selective detection of NFX and CFX, respectively, in human urine samples. Furthermore, molecular recognition of magnetic fluorescent sensor as well the corresponding computational modeling is in progress in our laboratory, which should spark a broad spectrum of interest due to its great versatility for future applications.
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Acknowledgment This work was supported by State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Natural Science Foundation of P. R. China (Item No. 61374218).
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Fig. 1 Models of the complexes formed from different numbers of CFX molecules and silanized APTES. (Si gray/blue; N blue; O red; C gray; H white; F green)
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Fig. 2 Schematic procedure of imprinting CFX in a silica matrix embedded with Fe3O4 nanoparticles and CdTe QDs.
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Fig. 3 HR-TEM images of CdTe QDs (a), TEM images of oleic acid capped Fe3O4 nanoparticles (b) and SEM image (c) and TEM image (d) of MIPs.
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Fig. 4 Fluorescence spectroscopy of the MIPs aqueous solution with the increasing concentrations of CFX adding in. Inset: Stern–Volmer-type description of the data showing a linear fit throughout the CFX concentration range.
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Table 1. Binding Energies (in KJ mol-1) of silanized APTES model with different numbers of CFX molecules.
Species CFX
silanized APTES Complex (a) Complex (b) Complex (c)
E –3013224.0990 –20237143.3304 –23250405.7616 –26263681.8455 –29276963.7054
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ΔE
– – –38.3322 –90.3172 –148.0782
