17β-estradiol (E2) is one of the several naturally occurring estrogens, which possesses a series of threats to wildlife and humans   . Due to its persistence and accumulation in the environment, numerous methods have been developed to decontaminate E2, such as adsorption , catalytic degradation   oxidation  and biodegradation    . Among which adsorption is a commonly adapted approach due to its relatively convenience and efficiency.
However, absorbent materials have been reported are still suffering drawbacks of slow adsorption rate, non-specific adsorption and complicated preparation process etc.
Silica nanoparticles have attracted great attention for their low toxicity, excellent chemical stability, and moderately modifiable surface  . The large surface area and functionalized hydroxyl groups on particle surface underlie facile modification and high loading effect of various absorbents  , which allows us to tune the scales and endow them with novel properties.
Molecular imprinting has been widely used for the development of tailor-made receptor binding sites in a three-dimensional, cross-linked polymer matrix. Molecularly imprinted polymers (MIPs) are thus generated with ligand-specific recognition properties analogous to biological systems, and have gained considerable attention in applications, such as solid-phase extraction   , chiral compounds isolation  , sensors  catalysis   and synthetic auxiliary  . Despite its wide applications and numerous fabrication methods of the conventional bulk MIPs, it still suffers some intrinsic limitations: the deep buried binding sites in bulk hinder subsequent template removal and the guest molecule diffusion process, thereby decreasing the imprinting efficiency; the mechanically crushing and grinding can cause the destruction of imprinted sites and cavities, resulting in the irregular shape, inhomogeneous imprinting sites and eventually poor site accessibility and low adsorption capacity to the guest molecule. In response to these intrinsic drawbacks, the surface imprinting technique has been proposed by creating the imprinted cavities with high affinity on the surface of a suitable matrix, which not only possesses high adsorption capacity but also avoids problems of limited mass transfer and template removal.
Several methodologies are adapted to prepare such MIP films include spin-coating  and surface-initiated atom transfer radical polymerization (ATRP)  . One major issue with these approaches is controlling the MIP film thickness while ensuring the quantity and quality of imprinting sites. Therefore, finding a facile way to fabricate robust and efficient nanostructured molecular imprinting systems is still greatly needed.
As is well known, various analytical methods have been applied in estrogen detection such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA) and some other analytical tools. Although some of these analytical techniques are widely used with good precision, their applications are seriously restricted due to the complicated and professional operation procedures, high cost and relative low sensitivity. Due to the excellent sensitivity, fast response, rich molecular information and non-destructive data acquisition, Raman is extensively used from biological analysis to environment monitoring and food safety, even clinical diagnosis and therapy. It could be a promising idea to combine MIPs with SERS for the selective adsorption and sensitive detection of estradiol.
In this work, we report a facile approach to prepare a highly-controllable core-shell silica-MIPs absorbent. The covalently modified templates along with KH-570 were grafted onto the surface of SiO2 nanoparticles and followed by polymerization. Thus, a MIPs layer was anchored on the surface of SiO2 nanoparticles via a surface molecular imprinting process. The performance of the as-synthesized materials towards E2 and their selectivity to structural analogues were investigated, which revealed that the core-shell MIP nanomaterial coupled with Raman, could be applied to the selective adsorption and sensitive detection of estradiol.
2.1. Reagents and Chemicals
All chemicals were of analytical grade reagents. Tetraethylorthosilicate (TEOS) was purchased from Gracia Chemical Technology Co., Ltd. Triethylamine (TEA), 3-methacryloxypropyltrimethoxysilane (KH-570), 3-isocyanatopropyltriethoxy-silane (IPTES) and acrylic acid (MAA) were supplied from Shanghai Aladdin Industrial Corporation. 17β-Estradiol (E2) was purchased from Adamas Reagent, Ltd. Ethylene glycol dimethyl acrylate (EGDMA) and 2,2'-Azobis (2 methylpropionitrile) (AIBN) were obtained from Guangdong J&K Scientific Ltd. Acetonitrile (ACN), tetrahydrofuran (THF), toluene were purchased from Sichuan Xilong Chemical Co., Ltd. Ammonia solution (25% - 28%), ethanol, methanol and acetone were obtained from Sichuan Kelong Chemical Co., Ltd. Ultrapure water (UPW) was used throughout the experimental procedures. Co., Ltd.
2.2. Preparation of SiO2 Nanoparticles (SiO2 NPs)
SiO2 NPs were prepared by the Stӧber method  . Ethanol (44 ml), water (11 mL), and ammonia solution (1.1 mL) were mixed in a flask and vigorously stirred at 40˚C. Then a mixing solution of TEOS (4.4 mL) and ethanol (20 mL) was quickly added to the above solution and the mixture was stirred for 15 h. The SiO2 NPs were centrifuged and rinsed with acetone. Finally, the obtained SiO2 NPs were dried at 120˚C for 24 h.
2.3. Anchoring of E2 and KH-570 on the Surface of SiO2 NPs
First, the template-functional monomer complex (EstSi) was prepared: E2 (1 mmol), IPTES (1.3 mmol), and TEA (8 mmol) were dissolved in THF. The mixture was stirred under N2 at 65˚C for 24 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by precipitation in n-hexane and filtration  . EstSi was collected as a white solid (70 mg).
SiO2-(E2+KH-570) were prepared by covalently attachment of KH-570 and EstSi to the surface of SiO2 nanoparticle. Firstly, the SiO2 NPs were dispersed in dry toluene (75 mL per gram of particles) under inert conditions. Then, EstSi (0.5 mmol per gram of particles) and KH-570 (1 mmol per gram of particles) were added to the suspension under constant stirring. The mixture was refluxed at 90˚C for 24 h. The resultant nanoparticles were centrifuged, filtered and washed several times with acetone. The solids were vacuum dried at 60˚C for 24 h to obtain 0.95 g of SiO2-(E2+KH-570). The SiO2-(IPTES+KH-570) were prepared by the same conditions, except that IPTES (0.5 mmol per gram of particles) was used instead of EstSi (0.5 mmol per gram of particles).
2.4. Preparation of 17β-Estradiol-Imprinted SiO2@MIP NPs
The above prepared SiO2-(E2+KH-570) (150 mg) were dispersed into 35 mL of THF under ultrasonication. MAA (0.1 mmol), EGDMA and AIBN (15 mg) were subsequently dissolved into this solution. The mixing solution was purged with nitrogen for 10 min. Polymerization was first done at 50˚C for 5 h, and subsequently at 60˚C for 24 h. The mixing solution was stirred at a rate of 300 rpm/min throughout the experiment. Then particles were collected by filtration and washed with acetone to remove the unreacted monomers and cross-linking agent, and dried under vacuum at The above prepared SiO2-(E2+KH-570) (150 mg) were dispersed into 35 mL of THF under ultrasonication. MAA (0.1 mmol), EGDMA and AIBN (15 mg) were dissolved into this solution. The mixing solution was purged with nitrogen for 10 min. Polymerization was first done at 50˚C for 5 h, and subsequently at 60˚C for 24 h. The mixing solution was stirred at a rate of 300 rpm/min throughout the experiment. Then particles were collected by filtration and washed with acetone to remove the unreacted monomers and cross-linking agent, and dried under vacuum at SiO2@MIP1-E2, SiO2@MIP2-E2, SiO2@MIP3-E2, respectively.
In order to remove the E2 templates in the imprinted shells, SiO2@MIP-E2 (220 mg) was refluxed in the solution of DMSO and water (5:1, v/v) at 180˚C for 3 h, and then the particles were obtained by Soxhlet extraction with the mixed methanol and acetic acid (8:2, v/v) solution at room temperature for 24 h. The resultant nanoparticles were centrifuged, filtered and washed several times with methanol. The solids were vacuum dried at 60˚C for 24 h. The samples obtained with different SiO2@MIP-E2 (SiO2@MIP1-E2, SiO2@MIP2-E2, SiO2@MIP3-E2) were denoted as SiO2@MIP1, SiO2@MIP2, SiO2@MIP3 respectively. Nonimprinted nanoparticles were prepared via the same procedure of SiO2@MIP2, but the SiO2-(IPTES+KH-570) (150 mg) was used in place of SiO2-(E2+KH-570), and assigned as SiO2@NIP1. Moreover, as a control, SiO2@MIP4 was also prepared in the same way as SiO2@MIP2 without adding MAA.
2.5. Adsorption Experiments of E2
The equilibrium adsorption experiments were performed to evaluate the binding capability of four kinds of SiO2@MIP (SiO2@MIP1, SiO2@MIP2, SiO2@MIP3, SiO2@MIP4) and SiO2@NIP1. Standard E2 solutions were prepared in acetonitrile and the E2 concentrations in the solutions were varied from 2 mg/L to 40 mg/L (2, 5, 10, 15, 20, 30, 40 mg/L). Then the 10 mg of SiO2@MIP or SiO2@NIP1 were dispersed into 10 mL of standard E2 solution. The obtained mixture was incubated at room temperature for 24 h, the suspension was filtered by the 0.1 µm micro-filter, and the filtered solution was analyzed by HPLC to obtain the E2 concentration.
In order to investigate the kinetics property of SiO2@MIP2, the adsorption kinetic experiments were performed as follows. E2 solutions were prepared in acetonitrile and the E2 concentration in the solution was 37 µmol/L. SiO2@MIP2 (50 mg) or SiO2@NIP1 (50 mg) were added into 10 mL of E2 solution. The obtained mixtures were conducted at room temperature for different contact time (5, 10, 15, 20, 30, 40, 60 min), respectively. The suspension was quickly filtered by the 0.1 µm micro-filter, and the filtrates were analyzed by HPLC to obtain the E2 concentration.
Selectivity experiments were conducted by using E2, Lupeol (LU), 3’,4’,5'-trimethoxyflavanone (TF) and 2-Hydroxynaphthalene (Np) as the structural analogues of E2. The 10 mg of SiO2@MIP2, SiO2@MIP4 and SiO2@NIP1 were dispersed into 2 ml of acetonitrile stock solution, which contains E2, LU, TF and Np at a concentration of 37 μmol/L, respectively. After incubating for 24 h at room temperature, the suspension was filtered by the 0.1 µm micro-filter, and the filtered solution was analyzed by HPLC to obtain the E2, LU, TF, Np concentration, respectively.
The reusability of SiO2@MIP2 was measured as below: standard E2 solution was prepared in acetonitrile and the E2 concentrations in the solution were 10 mg/L. The SiO2@MIP2 (100 mg) was dispersed into 20 mL of standard E2 solution. The mixture was incubated at room temperature for 24 h, the suspension was filtered by the 0.1 µm micro-filter. The filtrate was analyzed by HPLC. Then, the E2 of the recovered nanoparticles was removed with 10 mL of elution of methanol-acetic acid (8:2, v/v) for 24 hours to ensure complete removal of the E2 in the nanoparticles and washed with methanol, then vacuum dried at 60˚C for 24 h and reused for the adsorption of E2. The adsorption regeneration cycle was repeated five times with the same SiO2@MIP2.
2.6. Instruments and HPLC Analysis
Fourier transform infrared (FTIR) spectra were recorded with Perkin-Elmer system 2000 FT-IR spectrometer. 1H and 13C NMR spectra of EstSi were investigated by Bruker Ascend 400 MHz. Transmission electron microscopy (TEM) images were obtained on a Tecnai-G2-F20 electron microscope operating at 200 kV. Morphology of the materials were determined by scanning electron microscopy (SEM) with a JEOL 6300-F microscope.
The Raman spectra were collected on a HORIBA Lab Raman spectrometer (France) with an excitation wavelength of 532 nm, a resolution of 0.65 cm−1 and a beam diameter of 20 μm. Before Raman measurements, the SiO2@MIP2 (10 mg) or SiO2@NIP1 (10 mg) was incubated in the 2 ml of E2 acetonitrile solution (the E2 concentrations in the solution was 40 mg/L) for 24 h, then isolated by filtration, washed 3 times with acetonitrile and vacuum dried. The samples were denoted as SiO2@MIP2 rebinding E2 and SiO2@NIP1 rebinding E2, respectively. The Raman spectroscopy were recorded by focusing the 532 nm diode laser on the silica nanoparticle materials with a total exposure time of 300 s per spectrum, all spectrograms of samples were smoothed and the baselines were corrected.
A Shimazu Prominence LC-20 A HPLC (Kyoto, Japan) with an Acclaim 120-C18 column (Dionex, USA, 5 μm, 120 Å, 4.6 × 20 mm2). The UV detection wavelength was 208 nm, the flow rate was 1.0 mL∙min−1 and the injection volume was 25 µL. ACN/H2O (45/55, v/v) was used as the mobile phase.
3. Results and Discussion
The covalently modified estradiol-functional monomer (EstSi) was synthesized according to the Yang et al.’s method (Figure 1)  . SiO2 nanoparticles were synthesized according to reference  . The covalently modified templates (EstSi) along with KH-570 were grafted onto the surface of SiO2 nanoparticles and followed by polymerization to result in a MIPs layer on the surface of SiO2 nanoparticles  . The thickness of the MIPs layer was controlled by the added cross linker and the number of imprinted sites was controlled by EstSi used. The quality of the imprinted sites was controlled by both the covalently fixed template and coupling reagent (KH-570). The ratio of EstSi/KH-570 was optimized as 1:2. Finally, the template was removed by heating the SiO2@MIP-E2 in DMSO at 180˚C, followed by Sohlex extraction with a mixed solvent of methanol and acetic acid   . The preparation process was illustrated in Figure 2.
3.2. Characterization of the SiO2@MIP
The size and morphology of SiO2 nanoparticles were characterization by SEM (Figure 3). The as-synthesized SiO2 nanoparticles were uniform in both size and
Figure 1. Synthesis of the template-functional monomer complex (EstSi).
Figure 2. Schematic demonstration of the core-shell imprinting process.
Figure 3. SEM images of SiO2 nanoparticle.
shape, with a mean diameter of about 100 nm and relatively mono-disperse. They were used for further grafting of EstSi and KH-570.
Fourier Transform Infrared (FTIR) spectroscopy was used to identify functional groups of KH-570 grafted on SiO2-(E2+KH-570) and polymerized core-shell SiO2@MIP (Figure 4). FT-IR spectra of SiO2-(E2+KH-570) shows the characteristic band of C=O stretching vibration in the 1700 cm−1 region, which indicates the presence of the coupling reagent (KH-570)  . After polymerization with EGDMA and MAA, a new peak at 1733 cm−1 appears, which could be assigned to the C=O stretching (Figure 4, SiO2@MIP2), which verified the successful introduction of the functional groups onto the surfaces of the modified SiO2 nanoparticles by copolymerization   .
TEM images were obtained for SiO2, SiO2@MIP1, SiO2@MIP2 and SiO2@MIP3 to identify the core-shell structure (Figure 5). TEM images show that the thickness of the polymer layer of SiO2@MIP1 (Figure 5(b)), SiO2@MIP2 (Figure 5(c)), SiO2@MIP3 (Figure 5(d)) was about 1.0 ± 0.2 nm, 2.0 ± 0.8 nm, 5.0 ± 2.0 nm, respectively. These results indicate the thickness of the polymer shell depends on the amount of EGDMA used.
Raman spectra were used to confirm the incorporation of the template molecules (E2) in the SiO2@MIP2-E2 and removal of E2 from SiO2@MIP2. As shown in Figure 6, the SiO2@MIP2-E2 exhibited strong characteristic Raman bands of SiO2@MIP2-E2 around 829 cm−1, 930 cm−1, 1450 cm−1 (Figure 6(b)), indicating the successful incorporation of E2 in SiO2@MIP2-E2 (Figure 6(a) and Figure 6(b)). The disappearance of the typical signals of E2 at 829 cm−1, 930 cm−1, 1450 cm−1 from Figure 6(c), implicating that E2 was indeed removed from the polymer shell of SiO2@MIP2. The main bands of these Raman spectra and their assignments are shown in Table 1  .
3.3. Binding Property Investigation of the SiO2@MIP
The adsorption capacity was calculated according to the following equation:
Q = (C0 - Cf) V/M
Figure 4. FT-IR spectra of nanoparticles: (a) The pure SiO2 nanoparticles; (b) SiO2-(E2+KH-570); (c) SiO2@MIP2.
Figure 5. TEM images of nanoparticles: (a) pure SiO2 nanoparticles; (b) SiO2@MIP1; (c) SiO2@MIP2; (d) SiO2@MIP3.
Figure 6. Raman spectroscopy of: (a) E2 standard sample; (b) SiO2@MIP2-E2; (c) SiO2@MIP2.
Table 1. Raman spectral assignment for the E2 solid.
ν―stretching vibration; δ―internal deformation vibration (bending); the schematic diagram of the E2 molecule in Figure 6.
where C0 (mmol/L) is the initial con-centration of E2, Cf (mmol/L) is the concentration of E2 in the filtrate, V (mL) is the volume of the adsorption mixture, and M (mg) is the mass of SiO2@MIP2.
The isotherm of imprinted materials with added acrylic acid with shell thickness of 1 nm, 2 nm and 5 nm (Figure 7(b) SiO2@MIP1, Figure 7(c) SiO2@MIP2 and Figure 7(d) SiO2@MIP3), without acrylic acid of shell thickness of 2 nm (Figure 7(e) SiO2@MIP4), and non-imprinted material (Figure 7(a) SiO2@NIP) are presented in Figure 7, which clearly demonstrate that the material with added acrylic acid of imprinted shell thickness of 2 nm gave the highest adsorption of E2 (Figure 7(c)). The one without added acrylic acid (Figure 7(e)) otherwise identical with c showed a slightly lower adsorption than c. The shell thickness thicker or thinner than 2 nm showed poorer adsorption. The non-imprinted material had very low adsorption capacity for E2, due to non-specific adsorption. Thus, the material with added acrylic acid with thickness of 2 nm was used for further investigation in the adsorption kinetics and selectivity.
The core-shell imprinted material showed fast adsorption as demonstrated in Figure 8. The adsorption capacity of SiO2@MIP2 increased quickly at the first 5 mins and reached equilibrium after 10 mins. For SiO2@NIP1, the equilibrium time was 15 mins and the binding capacity was much lower than SiO2@MIP2. This phenomenon can be explained by that comparing with the SiO2@NIP1, SiO2@MIP2 have the specific binding sites for E2. The rapidly rebinding kinetics suggested SiO2@MIP2 has great potential in ultrafast enrichment and separation of E2 molecules.
The detection limit of the as-synthesized material was further tested with the Raman intensity changes after adding different concentrations of E2, as exhibited in Figure 9. The peaks at 829, 930 and 1450 cm−1 were selected for identifying E2. As the E2 concentration increased from 0.01 μmol/L to 1 μmol/L , the peak signals intensity slightly increased, with the lowest detectable concentration of 0.01 μmol/L , which was sufficient to meet the requirements of detection limit of E2 requirements in the environmental water samples.
The imprinting effects were further evaluated by adsorption specificity experiment for the template and its structural analogues, LU, TF and Np whose chemical structural formulae are shown in Figure10(a). The results were summarized in Figure 10(b). The adsoption ability of the non-imprinted material SiO2@NIP1 for E2 was much lower than the SiO2@MIPs, and there was no significant difference in
Figure 7. Adsorption isotherms: (a) SiO2@NIP1; (b) SiO2@MIP1; (c) SiO2@MIP2; (d) SiO2@MIP3; (e) SiO2@MIP4.
Figure 8. Kinetic curves of SiO2@MIP2 and SiO2@NIP1.
binding capacity for the competitive compounds.
The reusability of the materials is an important factor to improve the economic efficiency and extend practical uses. The adsorption-desorption cycles were repeated five times by regenerating the used imprinted. The reusability was evaluated according to the following equation:
R = Qn/Q0 × 100%
where R is regeneration efficiencies, the Qn (μg/g) is the adsorption capacity of
Figure 9. Raman spectra of SiO2@MIP2 incubated with E2 acetonitrile solution at concentration of (a) 1 μmol/L; (b) 0.5 μmol/L; (c) 0.1 μmol/L; (d) 0.05 μmol/L; (e) 0.025 μmol/L; (f) 0.01 μmol/L, respectively.
Figure 10. (a) The structure of the compounds used in the selectivity adsorption; (b) Selectivity adsorption result of four kinds of molecularly imprinted materials for E2, LU, Np and TF.
Figure 11. Reusability of the SiO2@MIP2 material.
regenerative experiments, the “n” is repeated times, Q0 (μg/g) is the adsorption capacity of original material.
As observed in Figure 11, calculations showed that of the prepared SiO2@MIP2 after using for 5 times, the absorption capacity was reduced by about 10.6%. The SiO2@MIP2 were repeatedly used and regenerated for 5 times with no significant loss of its original adsorb ability and selectivity. SiO2@MIP2 as a recyclable economic material has a potential applying prospect in the extraction, separation and concentration and trace analysis of 17β-estradiol. It is because that the appropriate thickness and the high stability of the polymer layer, and monolayer recognition sites are distributed uniformly on the surface of silica nanoparticles all can enhance reusability.
In summary, we have developed a core-shell silica-MIPs absorbent through anchoring a MIPs layer to the surface of SiO2 nanoparticles via a surface molecular imprinting process. The quantity and quality of imprinted sites on the surface of silica nanoparticle were ensured by covalently attached template precursors and coupling reagent KH-570 on SiO2 before polymerized imprinting. This material, combining with Raman spectra as detection method, can fast and selectively identify E2 among structural analogues, with detection limit of 0.01 μmol/L. This method can be adapted for other environmental contaminates or biological significant molecules detection by simply switch the template in the shell imprinting process.
Financial supports from Science and Technology Department of Sichuan Province (2016JY0034, 2017SZ0075) is gratefully acknowledged.
*These authors contributed equally to this work.