白藜芦醇分子印迹聚合物微球的制备及特性评价
0 IntroductionMolecularly imprinted polymers (MIPs) have been tailor-made materials with high selectivityfor a target molecule, widely used in solid-phase separation, membrane separation, environmentalapplication, and reaction catalysis[1-4]. Convention MIPs have been prepared in the form of bulkmonolith[5,6]. The copolymers are then ground and sieved to obtain appropriate size particles withirregularly shape for further use. This procedure is time-consuming and results in only moderateamounts of useful product. Lots of efforts have been made to prepare MIPs with regular size andshape for wide applications[7]. Recently, molecular imprinted polymeric microspheres (MIPMs)have been received much concern due to their potentional applications, which can be prepared byemulsion polymerization[8], suspension polymerization[9], precipitation polymerization[10],Multi-step swelling and polymerization[11], and surface molecular imprinting[12]. Of course,various methods have their own advantages and disadvantages. Compared with the abovementioned methods, a single-step swelling and polymerization might be the easiest methodsbecause this procedure is facile and the obtained MIPMs have good monodispersity.
Chloroform and cyclohexcanol are usually used as solvent for a single-step swelling andpolymerization[13,14], however, most template molecule such as active herbal ingredients,environmental pollutants and pesticides are not soluble in above-mentioned non-polar solvents.
Thus, it will be very interesting to prepare MIPMs in polar solvent system, suitable to polartemplate molecule.
Resveratrol is an interesting compound found in berries, wine, peanut and grapes. Thisnatural phytoalexin is expressed in plants in response to fungal infections and stress factorssuch as nutrient deprivation. Recent studies have shown that it has a variety of biologicalproperties, such as anti-inflammatory, immunomodulatory, cardioprotective and chemopreventiveactivities, and resveratrol has also been shown to extend the lifespan of yeast calls[15-17].
In this study, MIPMs have been prepared by a single-step swelling and polymerization usingpolystyrene as seeds in polar solvent. The factors of preparing microsphere have been studied indetail; it was found that variation of the swelling time and the solvent polarity played vital role oncontrolling morphology of microspheres. It was found that the adsorption rate of MIPMs was veryfast, due to their unique groove-style spatial structure.
1 Experimantal
1.1 Materials
Resveratrol (TCM038-080715, purity>98%) was from Nanjing Institute of Chinese MateriaMedica. Polydain (08071022, purity>98%) was from Shanghai Tauto Biotech Company. Waterwas deionized and distilled. All other chemicals and solvents were obtained from commercialsources with AR grade. Styrene was distilled to remove inhibitor before use. All other materialswere used without further purification.
1.2 Preparation of polystyrene seeds
Polystyrene seeds were synthesized as follows. 21.24 g styrene, 0.29 g sodium chloride and0.14 g potassium peroxodisulfate were added to 160 mL of ultra-pure water. The polymerizationwas carried out under nitrogen atmosphere at 70 ℃ for 20 h with magnetic stirring at 380 rmp.After purification by repeated centrifugation and dispersion, the purified seed was dispersed intowater to reach a final particle concentration of 0.2 g/mL for further use.
1.3 Preparation of MIPMs
In a 100 mL there-necked flask, 1 mmol resveratrol, 4 mmol acrylamide, 20 mmol ethyleneglycol dimethacrylate, 1.00 g dibutyl phthalate, 0.20 g azobisisobutyronitrile, and 5.00 g N,N-dimethylformamide (DMF) were mixed with 50 mL aqueous solution of 0.1 % (w/w) sodiumdodecanesulphonate and 1.0 % (w/w) polyvinyl alcohol, emulsified under ultrasonic conditionsuntil the size of oil drops became, at most, 0.5 μm (observed by optical microscope), then 1.5 mLpolystyrene seed dispersed in water was added dropwise, the mixture was stirred at roomtemperature until the emulsified organic phase was completely absorbed by the seed particles. Themixture was degassed with nitrogen for 20 min. The polymerization was carried out at 70 ℃ for20 h with continuous stirring. The obtained microspheres were washed with water, methanol andtetrahydrofuran. Then the micropheres were eluted by the mixed solvent of methanol and aceticacid (9:1, v/v) for several times to extract the template molecules until no resveratrol could bedetected by UV spectrometer in the eluent. Finally, the obtained micropheres were rinsed withethanol for one time to remove the remaining acetic acid, and dried in the vacuum desiccators for24 h before used. As a control, the non-molecularly imprinted polymer microspheres (NMIPMs)were prepared and treated in the same way, except that the template molecule was omitted fromthe polymerization process.
1.4 Binding experiments
Ten milligrams MIPMs or NMIPMs were added into 10 mL tubes, and mixed with 2.0 mL ofresveratrol-DMF solution with specific initial concentration ranging from 0 to 5.0 mmolL-1. Afterthe samples were shaken at 25 ℃ for 40 min, the solution was centrifuged at 4000 rpm for 15 min.
The concentration of free resveratrol in the supernate was measured by UV spectrophotometry at306 nm. The amount of resveratrol bound to the MIPMs was calculated by subtracting the amountof free resveratrol from the amount of resveratrol initially added. Meanwhile, the adsorptionkinetics of the MIPMs was performed at the different adsorption time intervals.
2 Results and discussion
2.1 Preparation of polystyrene microspheres
Polystyrene seeds were prepared by emulsifier-free polymerization. The morphology and sizewere characterized by scanning electronic microscopy (SEM) with a JEOL JSM-5610LV scanningelectronic microscope. Fig. 1 indicated the resulting polystyrene were monodisperse and uniformspherical morphology with about 2 μm in diameter.
2.2 Preparation of MIPMs
The SEM images indicated that MIPs prepared in DMF system were spherical structure asshown in Fig. 2. The microspheres were uniform and had good dispersibility with 5 μm indiameter. It was excited to find that the obtained microspheres had lots of concave-dipression orcracks.
2.2.1 Effect of solvent polarity on microsphere morphology
In order to examine the effect of the solvent, tetrahydrofuran and acetonitrile were used assolvent and other conditions were kept unchanged. It could be seen that the MIPMs prepared indifferent solvent system had almost the same size with about 5μm in diameter, whereas MIPMsprepared in N, N-dimethylformamide system had a big hole running through micropheres, andMIPMs prepared in acetonitrile system had deep cracks as shown in Fig. 3. That is to say, themorphologies of MIPMs could be controlled by changing the polarity of solvent.
2.2.2 Effect of swelling time on microsphere morphology
It was also found that the swelling time played vital role on the morphology of MIPMs. Afew polystyrene seeds could be seen and the size of MIPMs was not uniform as shown in Fig. 4A,when swelling time was 2 h. Some ruptured MIPMs could be seen as shown in Fig. 4B, when theswelling time increased up to 6 h.
2.3 Adsorption kinetics of MIPMs
The adsorption kinetics of the MIPMs for template molecule was carried out by adding 10mg MIPMs into 2 mL DMF solution with template molecule concentration of 1.5 mmol/L. Thecurve of the adsorption kinetics was shown in Fig. 5. It can be seen that the adsorption amount ofresveratrol increased with the increase of adsorption time. In the early 20 min, the adsorption rateincreased quickly. After 30 min, the adsorption almost reached equilibrium, indicating that theimprinted cavities were saturated with the template molecules. It was worthy mentioning theadsorption equilibrium time was much shorter [18-20]. In this communication, it was suggested theconcave-dipression spatial structure played vital role on the short equilibrium time. In thisstructure, the template molecules were easy to reach the imprinted cavities of the MIPMs. Thus,the adsorption rate was faster.
2.4 Binding property of MIPMs
The adsorption capacity is an important factor, which determines the amount of adsorbent fora given solution. The range of resveratrol solution with the concentration of 0-5 mmol was studied.
As can be seen in Fig. 6, the amount of resveratrol adsorbed per unit mass of MIPMs increasedwith the initial concentrations of resveratrol.
Where Kd is an equilibrium dissociation constant, Qmax is an apparent maximum number ofbinding sites. Q is MIPMs adsorption capacity for resveratrol (μmol/g) and C0 is quantitativelyconcentrate from resveratrol solution.
As shown in Fig. 7, Scatchard curve was a good straight line so that the MIPMs recognizingresveratrol only had one equivalent binding sites. The linear equation was as follows.
y=20.092-0.21053x, R=0.99508The apparent maximum number Qmax of binding sites is 95.435 μmol/g, nearly twice as manyas literature (48.50 μmol/g)[15]. It was speculated the larger adsorption capacities may be resultedfrom their unique concave-dipression structure, which effectively reduced the binding sites buriedin the MIPMs. Thus, there were more binding sites to be utilized.
2.5 Recognition properties of MIPMs
In order to verify the selectivity of the MIPMs for resveratrol, polydatin was used ascompetitive substrate. The reason was that polydatin are also the active ingredients of Polygonumcuspidatum Sieb. Et Zucc., and has similar structure with resveratrol. The molecular structures ofresveratrol and polydatin are shown in scheme 1. The selectivity of the polymers was estimated byKd and α same as Zhengs reports[14]. The obtained data was listed in Table 1.
The separation factor α was 1.74 indicated the MIPMs exhibited specific selectivity forresveratrol. That is to say, the template molecule could be easily seperated from its analogues bythe obtained MIPMs.
3 Conclusions
The single-step swelling and polymerization was developed to synthesize resveratrol MIPMsin polar solvent. Here, the unique concave-dipression spatial structure has successfully beenprepared using DMF as the solvent. The obtained MIPMs possessed the fast adsorption kineticsand large adsorption capacities. The MIPMs exhibit good recognition properties for theresveratrol.
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