Figure 3-1 displays the flow chart of the experimental design for preparation of sol-gel-derived E2 imprinted silica in this research. The preparation conditions including porogen, functional monomers, cross-linkers, TEOS/MTMOS, catalysts, pH values, and water/Si ratios were optimized to obtain the highest rebinding ability. Moreover, the compositions and textures of the MIPs were characterized by gas adsorption, Fourier transform infrared spectrometer (FTIR), and thermogravimetric analysis (TGA) to elucidate the effect of imprinting cavities and functional monomers on the adsorption behaviors.
Finally, the adsorptions of analogues were examined to understand the recognition ability of MIPs toward to the target compound
3-1 Chemicals
All reagents used in this study were commercially available and were used without further purifications. Tetraethyl orthosilane (TEOS, (C2H5O)4Si, Fluka, 99%) was used as the precursor of silica and the cross-linker in sol-gel process. 3-aminopropyltriethoxysilane (APTES, H2N(CH2)3SiOC2H5)3, Sigma-Aldrich, 99%) and phenyltrimethoxysilane (PTMOS, C6H5Si(OCH3)3, Sigma-Aldrich, 97%) were used as the functional monomers which bound the target compound 17β-estradiol (E2, C18H24O2, Alfa Aesar, 97%) via hydrogen bond and π-π stacking interactions, respectively. Methyltrimethoxysilane (MTMOS, C4H12SiO3, Sigma-Aldrich, 98%) was employed to increase the hydrophobicity and flexibility of polymers. Nonylphenol (C15H24O, Riedel-de Haën, 99.9%), 1-naphthaol (C10H8O, Riedel-de Haën, 99%), progesterone (C21H30O2, TCI), and testosterone (C19H28O2, TCI) were used in selective adsorptions. Absolute ethanol (C2H5OH, Sigma-Aldrich, HPLC grade, 99.8%), acetonitrile (C2H3N, Echo, HPLC grade, 99.9%), tetrahydrofuran
(C4H8O, Mallinckrodt, HPLC grade, 99.9%) were employed as porogens. Hydrochloric acid (HCl, Hanawa, 35%) and acetic acid (HAc, C2H4O2, Scharlau, 99.8%) were employed for catalysts test. In contrast to non-covalent bonding, 3-isocyanatopropyltriethoxysilane (ICPS, C10H21NO4Si, Sigma-Aldrich, 95%) was used to prepare monomer-template complex (E2Si) with E2 via covalent bond using dibutyltin dilaurate (DBDU, C32H64O4Sn, Sigma-Aldrich, 95%) as the catalyst. Dimethyl sulfoxide (DMSO, C2H6OS, Sigma-Aldrich, GC grade, 99.7%) was used as the extraction solvent for the covalent MIPs. Furthermore, toluene (C6H5CH3, J.T. Baker, HPLC grade, 99.9%) and methyl alcohol (CH3OH, Mallinckrodt, HPLC grade, 99.9%) were employed as adsorption solvent in this study.
Deionized water (DI, Millipore, 18 MΩ cm) was obtained from Milli-Q water purification system used throughout the experiments. Table 3-1 shows the structures of the major reagents used in the imprinted polymers.
Figure 3- 1. Flow chart of experimental design in this study.
Table 3- 1. The structure of the major reagents used in imprinted polymers.
Reagents Name Abbr. Structure
Template 17β-estradiol ( Target compound)
E2
O H
C OH
Cross-linker Tetraethylorthosilicate TEOS
Si O
Monomers Phenyltrimethoxysilane (for non-covalent)
3-2-1 Non-covalent MIPs
Molecularly imprinted organic-inorganic materials were produced by a sol-gel process.
Figure 3-2 shows the preparation process of sol-gel-derived MIPs. A 2.26 mL of TEOS together with 0.145 mL of MTMOS, and 5 mL of ethanol were mixed thoroughly at 200 rpm
in a 20 mL sample vial at room temperature. A 0.99 mL of DI and 0.12 mL HCl (0.1M) were added to the precursor solution slowly and mixed at room temperature for 30 min, then kept at 80 oC for 1.5 hr to hydrolyze and yield the sol. The homogeneous solution contained 5 mL of ethanol, 0.18 mL of PTMOS, 0.24 mL of APTES, and 0.14 g of E2 at a molar ratio of TEOS:PTMOS:APTES:E2=20:2:2:2:1 were added into the above solution. The mixture was stirred vigorously at 200 rpm at 80 oC for 2 hr. Then, condensation reaction was processed in the open vial at 60 oC in oven for 48 hr until gelation. The solution turned gradually from transparent to opaque white gel during the solvent evaporation. When the synthesis was completed, the gel monolith was dried at 100 oC for 6 hr in oven to evaporate the residue water and solvent. The gel was then crushed with a mortar and pestle into fine powders. A non-imprinted gel (NIP) was prepared using the same procedures except the addition of template (E2).
The crushed powders were immersed in 40 mL of hot methanol at 85 oC for 4 hr under stirring to remove the template. Washing was repeated 16 times until no trace of E2 could be detected. Further, the powders were washed by 20 mL fresh acetonitrile for 2 times and were dried at 70 oC.
Figure 3- 2. Preparation process of molecular imprinted organic-inorganic materials for E2.
3-2-2 Covalent MIPs
For comparison, covalent MIPs were prepared using ICPS as the functional monomer.
The ICPS was initially reacted with the phenol group of E2 to form urethane bond in the presence of DBDU. Figure 3-3 shows the mechanism of the reaction. A 0.14 g of E2 was dissolved in 5 mL of tetrahydrofuran with stirring at 200 rpm for 5 min. Then, 0.262 mL of ICPS and 0.05 mL of DBDU were added slowly into the E2 solution and underwent the reaction at 100 oC for 24 hr under nitrogen atmosphere.43, 53-55 The solution was called solution A.
On the other hand, 2.26 mL of TEOS, 0.145 mL of MTMOS, and 5 mL of tetrahydrofuran were mixed thoroughly at 200 rpm in a 20 mL sample vial at room temperature to form the precursor solution. A 0.99 mL of DI and 0.12 mL HCl (0.1M) were added to the precursor solution slowly and mixed at room temperature for 30 min and then kept at 80 oC for 1.5 hr to hydrolysis and yield the sol. Afterwards, the solution A and 0.18 mL of PTMOS were added into above solution. The mixture was stirred vigorously at 200 rpm at 80 oC for 2 hr. Then, condensation reaction was processed in the open vial at 60 oC in oven for 48 hr till gelation. When the synthesis was completed, the gel monolith was dried at 100 oC for 6 hr and crushed with a mortar and pestle into fine powders.
The template was extracted from the silica matrix by heating in a DMSO/DI (100 mL/20mL) solution at 190 oC for 8hr.53, 54 In the presence water, the isocyanato group was dissociated and converted to an amino group. The extraction mechanism of covalent MIPs was showed in Figure 3-4. Further, the powders were washed by 20 mL fresh aceonitrile for 2 times and were dried at 70 oC.
O
Figure 3- 3. Formation of urethane bond between E2 and ICPS.
Si N
3-3 Characterization
3-3-1 Specific Surface Areas (BET)
The specific surface areas and pore volume of the hybrid materials were analyzed by N2
adsorption technique and calculated using Brunauer-Emmett-Teller (BET) analysis. The 0.15 g of sample was degassed at 130 oC for 8 hr before analysis. Nitrogen gas physisorption and desorption was measured at 77 k under variety of relative pressure (p/po) (Micromeritics, Tri Star 3000). This method is based on the determination of quantity of nitrogen necessary to completely cover the surface of sample. The trapped amount of N2
gas was further applied to determine the total pore volume, which corresponded to the sum of the micropore and mesopore volume of samples. Relevant information of the average pore diameter could be obtained from the data of specific surface areas and total pore volume. In addition, each layer of adsorbate is treated as a Langmuir monolayer.
3-3-2 Fourier Transform Infrared Spectrometer (FTIR)
The Fourier transform infrared spectrometer (FTIR, Horiba, FT-730) was used to characterize the functional groups of polymers between 400 and 4000 wavenumbers (cm-1).
The samples were diluted with KBr and were pressed as self-standing pellets. The spectra were recorded with a resolution of 4 cm-1 for 100 times when pure KBr was employed as background.
3-3-3 Thermogravimetric Analysis (TGA)
The thermal property of MIPs was examined by a thermogravimetric analysis (TGA, TA instrument, Q500) to understand the changes of mass loss with the elevation of temperature.
Measurements were taking from room temperature to 700 oC at 10 oC/min under air flow of 60 mL/min.
3-4 Adsorption
50 mg of particles were mixed with 5 mL E2/acetonitrile solution (150 mg/L) and shaker at room temperature for 4 hr. The solution was then centrifuged at 14000 rpm for 10 min.
The adsorption capacity was determined by the reduced amount of E2 in the supernatant using a UV spectrometer (HITACHI U-3010). The absorption at 200-330 nm was recorded and the changes in absorption intensity at 280 nm were measured to calculate the concentration of E2. The amount of adsorbed E2 was calculated to obtain the adsorption capacity (Kd) of the MIPs by equation 3-1.
Where Co (mg/L) and Ce (mg/L) represented initial and equilibration concentration of E2, V (L) was the volume of solution, and m (g) was the weight of the imprinted polymers.
The imprinted factor (I) was calculated using equation 3-2.
( )
Where Kd (MIPs)and Kd (NIP)are the adsorption capacity of E2 on molecular imprinted and control polymer, respectively.
3-4-1 Equilibrium
Adsorption equilibrium of MIPs and related NIP toward E2 were determined by measuring the adsorbed amount of E2 in series sampling tubes at varies time intervals. 50
mg of samples were added to 5 mL of E2/acetonitrile solution (150 mg/L) in five tubes and mixed by shaker at room temperature. Then, each of them was centrifuged at 14000 rpm for 10 min to remove the polymers. The remaining concentrations of E2 in the supernatants were identified using UV-vis spectrometer.
3-4-2 pH effect
To understand the effect of pH values on the adsorption capacities, various pH (pH 3.2-7.2) were adjusted using HCl. The E2 was dissolved into acetonitrile and then added into HCl solutions (acetonitrile/DI=50/50, v/v) with different pH values. 25 mg of the MIPs were added to 5 mL of the 150 mg/L E2 solutions in test tubes when different pH values were used. The mixtures were shaken at room temperature for 4 hr and then centrifuged at 14000 rpm for 10 min. The reduction of the concentrations of E2 in the supernatants was analyzed using UV-vis spectrometer, and the rebinding capacity was calculated using the equation 3-1.
3-4-3 Solvents
To understand the effect of solvents on the adsorption capacities, various solvents including toluene, tetrahydrofuran, ethanol, acetonitrile, and methanol were used for the adsorption test. The E2 was dissolved into the solvents and then used for the rebinding tests.
25 mg of samples were added to 5 mL of 150 mg/L E2 of vary solvents in a centrifuge tube when different solvents were used. The mixtures were shaken at room temperature for 4 h and then centrifuged at 14000 rpm for 10 min. The concentration of E2 in the supernatant was determined based the intensity of its characteristic absorption and the rebinding capacities were calculated using the equation 3-1. The characteristic absorption changed a little bit in different solvents, and its appeared at 281.5 nm in ethanol, 280 nm for acetonitrile, 281 nm for methanol, 290.5 nm for tetrahydrofuran, and 288 nm for Toluene.
3-4-4 Selectivities
To understand the selective capability of MIPs for the target compound, analogues including nonylphenol, 1-naphthol, progesterone, and testosterone were selected to analyze the binding sites with E2. The selective adsorption of MIPs was carried out in individual and mixed solution when acetonitrile was used as the solvent. The physicochemical properties of E2 and its analogues were summarized in Table 3-2.
Table 3- 2. Physicochemical properties of compounds used for selective adsorptions test.
Compound Chemical structure Aqueous solubility Log Kow Ref.
17β-estradiol (E2)
O H
C OH 3.6 mg/L 4.01 76
Nonylphenol
O H
4.9 mg/L 4.48 76
1-naphthol OH 870 mg/L 2.85 77
Progesterone
C C
O
O C 27 mg/L 3.67 78
Testosterone C OH
O
23.4 mg/L 3.32 79
For individual compound adsorption, 25 mg of samples were added to 5 mL of 150 mg/L of E2, nonylphenol, and 1-naphthol, and 50 mg/L of E2, progesterone, and testosterone solutions individually and shaken at room temperature for 4 hr. Then, the suspensions were centrifuged at 14000 rpm for 10 min. The changed intensities of the characteristic absorptions of 1-naphthol, nonylphenol, progesterone, and testosterone at 323, 279, 237.5, and 238 nm respectively were taken to calculate their absorption capacities using the equation 3-1. For completive systems, the 1-naphthol, nonylphenol, and E2 compounds were dissolved in acetonitrile to reach 150 mg/L for each one. 25 mg of MIPs was added to 5 mL mixture solution and shaken at room temperature for 4 hr, and then centrifuged at 15000 rpm for 10 min. In order to remove the tiny suspending particles, the supernatant (3 mL) from the first centrifugation was transferred into another tube and underwent centrifugation at 15000 rpm for 20 min. Following 1 mL of the supernatant was analyzed by HPLC (Agilent Technologies 1200 series) to determine the adsorption capacity of imprinted polymers toward the three compounds. The compounds in mixtures were separated by a security guard column and an analytical reversed-phase column (Phenomenex LUNA, C18(2) column, 5 μm, 4.6×250 mm). The eluent was 100% acetonitrile at a flow rate of 1 mL/min. The injection volume was 50 μL, and the column effluent was monitored at 280 nm. The selectivity factor (S) was determined by the equation 3-3:
( 2)
( )
d E d compound
S K
= K
(3-3)
Where Kd (E2)and Kd (compound)are the adsorption capacity of MIP toward estradiol and tested compound.