Highly-Controllable Imprinted Polymer Nanoshell on the Surface of Silica Nanoparticles for Selective Adsorption of 17 β-Estradiol

A highly-controllable core-shell silica-MIPs absorbent by anchoring a MIPs layer to the surface of SiO2 nanoparticles via a surface molecular imprinting process was prepared. The templates were covalently modified with functional monomers to form precursor EstSi. The latter together with coupling reagent KH-570, were grafted onto the surface of SiO2 nanoparticles before polymerization, to ensure the quantity and quality of imprinted sites on the surface of the covalently attached matrix. The as-synthesized core-shell nanomaterials (SiO2@MIP2) were then evaluated for selective adsorption of 17β-estradiol (E2) with Raman spectra as detection method. The results indicate that SiO2@MIP2 can fast and selectively adsorb E2 from structural analogues, with detection limit of 0.01 μmol/l.


Introduction
17β-estradiol (E2) is one of the several naturally occurring estrogens, which possesses a series of threats to wildlife and humans [1] [2].Due to its persistence and accumulation in the environment, numerous methods have been developed to decontaminate E2, such as adsorption [3], catalytic degradation [4] [5] oxidation [6] and biodegradation [7] [8] [9].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 [10].The large surface area and functionalized hydroxyl groups on particle surface underlie facile modification and high loading effect of various absorbents [11] [12], 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 [13] [14] [15], chiral compounds isolation [16] [17], sensors [18] catalysis [19] [20] and synthetic auxiliary [21].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 [22] and surface-initiated atom transfer radical polymerization (ATRP) [23].
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 chro- 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 SiO 2 nanoparticles and followed by polymerization.Thus, a MIPs layer was anchored on the surface of SiO 2 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.

Preparation of SiO2 Nanoparticles (SiO2 NPs)
SiO 2 NPs were prepared by the Stӧber method [24].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 SiO 2 NPs were centrifuged and rinsed with acetone.Finally, the obtained SiO 2 NPs were dried at 120˚C for 24 h.

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 N 2 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 [25].EstSi was collected as a white solid (70 mg).

Preparation of 17β-Estradiol-Imprinted SiO2@MIP NPs
The above prepared SiO 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 SiO 2 @MIP1-E2, SiO 2 @MIP2-E2, SiO 2 @MIP3-E2, respectively.

Adsorption Experiments of E2
The equilibrium adsorption experiments were performed to evaluate the binding capability of four kinds of SiO 2 @MIP (SiO 2 @MIP1, SiO 2 @MIP2, SiO 2 @MIP3, SiO 2 @MIP4) and SiO 2 @NIP1.Standard E2 solutions were prepared in acetoni-Journal of Encapsulation and Adsorption Sciences trile 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 SiO 2 @MIP or SiO 2 @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 SiO 2 @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.SiO 2 @MIP2 (50 mg) or SiO 2 @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.
The reusability of SiO 2 @MIP2 was measured as below: standard E2 solution was prepared in acetonitrile and the E2 concentrations in the solution were 10 mg/L.The SiO 2 @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 SiO 2 @MIP2.

Instruments and HPLC Analysis
Fourier transform infrared (FTIR) spectra were recorded with Perkin-Elmer system 2000 FT-IR spectrometer. 1 H and 13 C 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 SiO 2 @MIP2 (10 mg) or SiO 2 @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 SiO 2 @MIP2 rebinding E2 and SiO 2 @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.

Preparation
The covalently modified estradiol-functional monomer (EstSi) was synthesized according to the Yang et al.'s method (Figure 1) [25].SiO 2 nanoparticles were synthesized according to reference [24].The covalently modified templates (Est-Si) along with KH-570 were grafted onto the surface of SiO 2 nanoparticles and followed by polymerization to result in a MIPs layer on the surface of SiO 2 nanoparticles [26].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 SiO 2 @MIP-E2 in DMSO at 180˚C, followed by Sohlex extraction with a mixed solvent of methanol and acetic acid [25] [26].The preparation process was illustrated in Figure 2.

Binding Property Investigation of the SiO2@MIP
The adsorption capacity was calculated according to the following equation:    where C 0 (mmol/L) is the initial con-centration of E2, C f (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 SiO 2 @MIP2.
The isotherm of imprinted materials with added acrylic acid with shell thickness of 1 nm, 2 nm and 5 nm ( 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 SiO 2 @MIP2 increased quickly at the first 5 mins and reached equilibrium after 10 mins.For SiO 2 @NIP1, the equilibrium time was 15 mins and the binding capacity was much lower than SiO 2 @MIP2.
This phenomenon can be explained by that comparing with the SiO 2 @NIP1, SiO 2 @MIP2 have the specific binding sites for E2.The rapidly rebinding kinetics suggested SiO 2 @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 SiO 2 @NIP1 for E2 was much lower than the SiO 2 @MIPs, and there was no significant difference in     regenerative experiments, the "n" is repeated times, Q 0 (μg/g) is the adsorption capacity of original material.
As observed in Figure 11, calculations showed that of the prepared SiO 2 @MIP2 after using for 5 times, the absorption capacity was reduced by about 10.6%.The SiO 2 @MIP2 were repeatedly used and regenerated for 5 times with no significant loss of its original adsorb ability and selectivity.SiO 2 @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.

Conclusion
In summary, we have developed a core-shell silica-MIPs absorbent through anchoring a MIPs layer to the surface of SiO 2 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 SiO 2 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.

X.
Fu et al.DOI: 10.4236/jeas.2018.84011211 Journal of Encapsulation and Adsorption Sciences matography-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 com-X.Fu et al.Journal of Encapsulation and Adsorption Sciences bine MIPs with SERS for the selective adsorption and sensitive detection of estradiol.

SiO 2 -
(E2+KH-570) were prepared by covalently attachment of KH-570 and EstSi to the surface of SiO 2 nanoparticle.Firstly, the SiO 2 NPs were dispersed in X. Fu et al.Journal of Encapsulation and Adsorption Sciences 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 SiO 2 -(E2+KH-570).The SiO 2 -(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 -(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 SiO 2 -(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

Figure 7 (
b) SiO 2 @MIP1, Figure7(c) SiO 2 @MIP2 and Figure7(d) SiO 2 @MIP3), without acrylic acid of shell thickness of 2 nm (Figure7(e) SiO 2 @MIP4), and non-imprinted material (Figure7(a) SiO 2 @NIP) are presented in Figure7, which clearly demonstrate that the material with added acrylic acid of imprinted shell thickness of 2 nm gave the highest adsorption of E2 (Figure7(c)).The one without added acrylic acid (Figure7(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.

Figure 10 .
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.

Table 1 .
Raman spectral assignment for the E2 solid.