Preparation of molecularly imprinted nanospheres by premix membrane emulsification technique

Journal of Membrane Science 417–418 (2012) 87–95

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Preparation of molecularly imprinted nanospheres by premix membrane emulsification technique Xing Kou a, Qiang Li a, Jiandu Lei a,n, Liyuan Geng a, Hongquan Deng b, Guifeng Zhang a, Guanghui Ma a,n, Zhiguo Su a, Qiying Jiang b a b

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China Engineering Research Center of Biomass Materials, Ministry of Education, Southwest University of Science and Technology, Mianyang 621010, China

a r t i c l e i n f o

abstract

Article history: Received 27 April 2012 Received in revised form 7 June 2012 Accepted 11 June 2012 Available online 21 June 2012

A novel method for the preparation of molecularly imprinted nanospheres is presented by premix membrane emulsification technique. On the example of chloramphenicol (CAP), the imprinted nanospheres were prepared using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EDMA) as cross-linker. The synthesis conditions of the nanospheres were optimized. When the membrane pore size is 1.4 mm, transmembrane pressure is 2.00 MPa, transmembrane numbers are 5 times and the volume ratios of oil–water in the coarse emulsions is 1:20, the resulted nanospheres are relatively uniform and their sizes are between 300–800 nm. Furthermore, the effect of the ratio of CAP–MAA on imprinting performance was investigated, and the selective adsorption experiment indicates that the imprinted nanospheres have good selectivity for CAP. Additionally, adsorption kinetics and adsorption isotherm of the imprinted nanospheres exhibit that the adsorption equilibrium time is around 60 min and the maximum theoretical static binding capacity is up to 158.78 mg g  1. Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved.

Keywords: Molecularly imprinted polymers Nanospheres Premix membrane emulsification Chloramphenicol Synthesis

1. Introduction Molecularly imprinted polymers (MIPs) have been demonstrated possessing unique and predetermined selectivity for target molecules [1–4]. To date, they have been widely developed for a variety of applications including separation [5], controlled release [6], solid-phase extraction [7], sensors [8], enzyme inhibitors [9] and antibodies [10]. The classical method for preparing MIPs was bulk polymerization because of its simplicity and universality [11]. However, the resulted imprinted polymers are block that need to be crushed and ground to obtain particles. These particles are irregular in size and shape and usually they contain a large portion of waste fine particulate material, which need additional sieving to obtain a narrow size distribution and remove fine particles. The whole process is tedious and time-consuming, and the resulted particles have low capacity and unfavorable site accessibility to the target [12]. Recently, some approaches for the preparation of spherical MIPs have been developed [13,14], the most widely used methods mainly include suspension polymerization [15,16] and precipitation polymerization [17–19]. For suspension polymerization, the

n

Corresponding authors. Tel.: þ 86 10 82544997; fax: þ 86 10 82544932. E-mail addresses: [email protected] (J. Lei), [email protected] (G. Ma).

beads obtained have the diameter which varies between 5 and 100 mm depending on the stirring speed and the amount of surfactant. The imprinted microspheres are polydisperse by this technique. Moreover, it is almost impossible to prepare the nanosized imprinting spheres by suspension polymerization. While, precipitation polymerization is a methodology which can prepare both micro- and nano-sized imprinting particles (0.3–10 mm), and the resulted particles are very uniform. Nevertheless, it needs a large excess of template molecules, and the particle productivity is low. In addition, the size of particles is difficult to be controlled, and the size distribution obtained by this method is significantly impacted by microenvironment, such as physical and chemical properties of template molecules, even different batch of solvents. Premix membrane emulsification is an attractive technique for the production of small sized and relatively monodisperse emulsions [20,21]. The process primarily includes two steps: (1) The coarse emulsions with big size and broad size distribution are formed using conventional emulsification methods; (2) The coarse emulsions are extruded through a membrane under higher pressure to produce uniformly sized nanodroplets. This technique can produce a homogenized emulsion, and the emulsion may be passed through the membrane repeatedly depending on the desired level of homogenization [22]. Premix membrane emulsification technique is simple and easy to be scale-up, which makes it become a promising technique for large-scale production of emulsions [23]. Up to now,

0376-7388/$ - see front matter Crown Copyright & 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.memsci.2012.06.023

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there is no report about molecularly imprinted particles using premix membrane emulsification technique. Chloramphenicol (CAP) is a broad-spectrum antibiotic exerting activity against a variety of bacteria through protein inhibition. However, it is proved that CAP has serious side-effects on humans in the form of bone marrow depression, and fatal aplastic anemia [24]. For these health concerns, CAP has been strictly banned for use in food-producing animals in many countries. Therefore, it is important to develop an effective and reproducible detection or extraction method to control and monitor CAP residues in food of animal origin. The preparation of molecularly imprinted nanospheres with premix membrane emulsification method is described in this study, in which CAP is used as template molecule and MAA is used as functional monomers. The operating factors such as transmembrane pressure, number of transmembrane cycles and volume ratio of oil–water phase were optimized in terms of their influence on emulsion properties. In addition, the imprinting performance of the imprinted nanospheres for CAP was evaluated including selectivity and adsorption capacity.

2. Materials and methods 2.1. Materials Erythromycin (EM), chloramphenicol (CAP), ethylene glycol dimethacrylate (EDMA) and florfenicol (FF) were obtained from Sigma-Aldrich (USA, AR). Methacrylic acid (MAA) was provided by West Long Chemical Plant (China, AR). Ethyl acetate, phosphoric acid, acetic acid and methanol were from Beijing Chemical Plant (China, AR). The free radical initiator, 2, 2-azobis (2-isobutyronitrile) (AIBN), was supplied by Shanghai Reagent fourth plants (China, AR). 2.2. Preparation of imprinted nanospheres using premix membrane emulsification The synthesis of molecular imprinted nanospheres with premix membrane emulsification technique was as follows: the template molecule CAP, the functional monomer MAA, the cross-linker EDMA and the initiator AIBN were dissolved in porogen solvent ethyl acetate as oil phase. Then they were saturated with dry nitrogen for 10 min, and added into water solution which contained a proportion of PVA. Then the mixture solution was homogenized at 24,000 rpm for 1 min to produce the coarse emulsion with larger size droplets. Then the coarse emulsions were extruded through the uniform pores of the Shirasu Porous Glass (SPG) membrane to obtain uniform smaller droplets (Fig. 1), which were further polymerized to achieve nanospheres at 60 1C for 24 h under stirring and N2 atmosphere. The obtained nanospheres were collected by centrifugation at 10,000 rpm

for 10 min, and washed respectively with hot water and methanol/ acetic acid solution (4:1, v:v) until no template molecule can be detected by spectrophotometer. Finally, they were washed with methanol and water, and then dried in a vacuum drier. Non-imprinted polymers (NIPs) nanospheres were synthesized under identical conditions in the absence of template molecules. Unless specified, the standard formulation conditions are as follows: the pore size of the membrane is 1.4 mm, the concentration of PVA is 1.5 wt%, the volume ratio of oil to the water phase is 1:20, the molar ratio of MAA to EDMA is 1:4. 2.3. Morphological characterization of the nanospheres Specific surface areas and porosity properties of MIPs/NIPs were measured by nitrogen sorption porosimetry (Micrometrics Instrument, ASAP 2020). MIPs and NIPs were degassed at 80 1C under nitrogen flow for 5 h prior to measurement, respectively. The specific surface areas were calculated by the Brunauer–Emmett– Teller (BET) equation [25], and the specific pore volumes were obtained in terms of the Barrett–Joyner–Halenda (BJH) theory [26]. The morphology of MIPs and NIPs were obtained by scanning electron microscopy (JSM-6700, JEOL). Prior to observation, the dry particles were attached to silver papers and then sprayed with gold. The average particle diameter was analyzed by zeta with analysis function for sub-micron particle size (Brookhaven Instruments Corporation, USA). 2.4. Binding experiments An amount of 30 mg MIPs or NIPs were weighed into 10 mL screw-cap vial and mixed with 6 mL methanol/water (5:95, v:v) solution of CAP. The sample was shaken for 3 h at 25 1C, and centrifuged at 10,000 rpm for 10 min. Then, the concentration of free CAP in supernatant was measured by UV spectrophotometer at 276 nm. According to the change of the concentration of CAP in solution before and after binding, the adsorption capacity value Q of nanospheres for CAP are calculated by Eq. (1), where C0 and C are respectively the initial and equilibrium concentration of CAP, V is the volume of the solution, W is the mass of nanospheres. Q ¼ ðC 0 CÞ  V=W

ð1Þ

2.5. Selectivity of the imprinted nanospheres In general, in order to evaluate the selective recognition ability, a few analytes with similar structure and properties as template molecules are used to perform the experiments. If the adsorption capacity of MIPs for template molecules is much bigger than these of structural similar compounds, the selectivity is good. In this study, two other kinds of antibiotics (EM and FF) were selected as competitive agents to estimate selectivity of the imprinted nanospheres for CAP. Structures of CAP, FF and EM are shown in Fig. 2. The MIPs or NIPs (30 mg) were placed respectively into six centrifuge tubes having 6 mL of methanol–water (5:95, v:v) solution of different substrate (EM, CAP or FF). These samples were stirred at 100 rpm and incubated for 3 h, and centrifuged at 10,000 rpm for 10 min. The free EM, CAP and FF in supernatant were measured respectively by UV spectrophotometer at 235 nm, 276 nm and 362 nm. 2.6. Adsorption kinetics of the imprinted nanospheres

Fig. 1. Schematic diagram of miniature kit for premix membrane emulsification.

The adsorption kinetics behavior of the imprinted nanospheres for CAP was studied by changing the adsorption time from 0 min to 270 min. The initial concentration of CAP was kept a constant

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O O

H 3C

CH3

O F

S

HO

O

OH CH3

H 3C

Cl N H

HO O

O CH3 CH3 O

N

O

Cl

OH

CH3

FF

O CH3 CH3

CH3 OH

OH

OH

OH O CH3 CH3

Cl O2N

HN Cl

CH3

O CAP

EM Fig. 2. Chemical structure of CAP, EM and FF.

of 100 mg mL  1 in methanol–water solution (5:95, v:v). Adding 30 mg of microspheres into 6 mL solution, then they were mixed at room temperature under stirring. After being absorbed for some time, each sample was centrifuged and 3 mL of supernatant was detected with UV–vis spectrophotometric analysis.

3. Results and discussion In this study, MIPs and NIPs nanospheres were synthesized by premix membrane emulsification technique, in which the water phase was a water solution containing a certain content of PVA, and the ethyl acetate solution containing CAP, MAA, EDMA and AIBN was used as the oil phase. In this polymerization system, MAA is used as functional monomer because in the structure of the template molecule CAP, there are HO–, –NO2, and –NH– functional groups, MAA not only can form electrostatic interaction with the basic functional group of CAP but also can interact with CAP by hydrogen bond. For premix membrane emulsification method, five factors play key roles during the preparation of uniform size imprinted nanospheres, including transmembrane pressure, transmembrane number (i.e. the number of circles that emulsions pass through a membrane), the PVA concentration in the water phase and the volume ratio of oil phase to water phase. Furthermore, one of the important factors that mainly affect the imprinting effect of imprinted nanospheres is the ratio of template CAP to the functional monomer MAA. Consequently, to obtain molecularly imprinted nanospheres for CAP possessing both uniform size and good imprinting performance, the following several different factors were studied in details. 3.1. Effect of transmembrane pressure on homogeneity of nanospheres In the absence of template CAP for the synthetic system, the nanospheres were prepared under different transmembrane pressures: 1.55, 1.70, 1.85 and 2.00 MPa. The other conditions were the same as indicated in Section 2.2. The scanning electron microscopy (SEM) images of nanospheres with different transmembrane pressure are shown in Fig. 3, and the corresponding size distribution is

displayed in Fig. 3(E). It suggests that the narrowest size distribution of nanospheres is achieved when the transmembrane pressure is 2.00 MPa. The reason may be that the higher pressure result in stronger collision between coarse emulsions and pore walls of SPG membrane, which causes stronger breaking of droplets. But, when transmembrane pressure is lower than 2.00 MPa, there is not enough power to break droplets into smaller droplets, which leads to many large droplets and poor uniformity. Moreover, due to the maximum pressure limit 2.00 MPa for this premix membrane emulsification equipment, 2.00 MPa is employed in the subsequent experiments. 3.2. Effect of transmembrane number on homogeneity of nanospheres The nanospheres were prepared by different transmembrane numbers: one, three and five. SEM images of nanospheres under different numbers of passes are shown in Fig. 4, and the corresponding size distribution is displayed in Fig. 4(D). It can be seen that the homogeneity of nanospheres increases and the size become smaller with the increase of transmembrane numbers. The larger droplets are broken into smaller droplets when each passes. The amount of larger droplets being broken into smaller ones increases with the increase of transmembrane numbers, which lead to smaller size and narrower size distribution of nanospheres. Therefore, the transmembrane number is selected as five times. 3.3. Effect of PVA amount in water phase on uniformity of nanospheres To stabilize emulsion, we use PVA as the stabilizer and the emulsifier. PVA is a kind of amphiphilic material. The hydroxyl groups in PVA will interact with the water phase while the vinyl chain will interact with ethyl acetate thus making the formed emulsion more stable. Thus, the addition of PVA will affect the stability of emulsion. Furthermore, in this case, PVA concentration will affect the size and size distribution of the emulsion. In this study, different concentrations of PVA, 1, 1.5 and 2 wt%, were utilized to prepare nanospheres. The transmembrane pressure and the transmembrane numbers are 2.00 MPa and 5 times,

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110 100

1.55MPa 1.70MPa 1.85MPa 2.00MPa

Intensity (%)

90 80 70 60 50 40 30 20

0

1000

2000 3000 4000 Particle diameter (nm)

5000

Fig. 3. SEM images of nanospheres prepared under different transmembrane pressure. (A) 1.55 MPa, (B) 1.70 MPa, (C) 1.85 MPa, (D) 2.0 MPa and (E) Size distribution of nanospheres prepared under different transmembrane pressure.

respectively. The other conditions are the same as described in Section 2.2. As shown in Fig. 5, the nanospheres with excellent homogeneity and narrow size distribution are obtained when the amount of PVA in the water phase is 1.5 wt%. On the one hand, it is difficult to prevent effectively the cohesion or coalescence among emulsion droplets each other when the PVA amount in water phase is below 1.5 wt%, which may results in larger size emulsion droplets and broader size distribution of emulsion droplets. On the other hand, when the concentration of PVA in the water phase is increased to be above 1.5 wt%, the interfacial tension between oil and water phase is further reduced, it could lead to the formation of lots of smaller droplets during the production of the coarse emulsion. And these smaller droplets pass through membrane pores easily without being broken into smaller ones, which can cause a wider size distribution.

transmembrane pressure, the transmembrane number and the PVA amount are 2.00 MPa, 5 times and 1.5 wt%, respectively. The other conditions are the same as described in Section 2.2. The SEM images of nanospheres obtained at different volume ratios of oil to water phase are shown in Fig. 6, and the corresponding size distribution is shown in Fig. 6(E). It can be seen that, nanospheres with better homogeneity and narrower size distribution are obtained when the volume ratio of oil to water is 1:20. When the ratio of oil to water phase is 1:13.3, the PVA concentration is high due to the reduction of water, thereby the amount of smaller droplets increases as indicated in Section 3.3. On the contrary, when the ratio of oil to water phase is 1:30 or 1:40, the PVA concentration in water is lower, which could result in many larger droplets. Therefore, the volume ratio of oil to water phase is fixed at 1:20 in the following experiments.

3.4. Effect of ratio of oil to water phase on uniformity of nanospheres

3.5. Effect of ratio of CAP to MAA on imprinting performance of nanospheres

The nanospheres were prepared under different ratios of oil to water phase: 1:13.3, 1:20, 1:30 and 1:40, in which the

The ratio of template molecule to functional monomer plays a key role for imprinting effects. In this case, three different molar ratios

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110 100

1 time 3 times 5 times

Intensity (%)

90 80 70 60 50 40 30 20

200

400

600

800

1000

1200

1400

Particle diameter (nm) Fig. 4. SEM images of nanospheres obtained under different number of transmembrane. (A) One, (B) Three, (C) Five and (D) Size distribution of nanospheres prepared under different number of transmembrane.

110 100

1.0% 1.5% 2.0%

Intensity (%)

90 80 70 60 50 40 30 20

200

400

600

800

1000

1200

1400

Particle diameter (nm) Fig. 5. SEM images of nanospheres prepared at different concentrations of PVA in the water phase. (A)1 wt%, (B) 1.5 wt%, (C) 2 wt% and (D) Size distribution of nanospheres prepared at different concentrations of PVA.

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110 100

1:13.3 1:20.0 1:30.0 1:40.0

Intensity (%)

90 80 70 60 50 40 30

20 100 200 300 400 500 600 700 800 900 1000 Particle diameter (nm)

of template molecule to functional monomer were investigated in order to obtain the imprinted nanospheres with excellent imprinting effect. Moreover, one commonly used molar ratio of template molecule to crosslinker EDMA (1:20) was adopted in this work [27]. As shown in Fig. 7, when the molar ratio of CAP to MAA is 1:2, the imprinted nanospheres prepared have the strongest adsorption ability to template molecule. When the molar ratio of template molecule to functional monomer is 1:1, the amount of functional monomer MAA is not enough, which results in less imprinting sites. On the other hand, when the molar ratio of CAP to MAA is 1:4, the amount of functional monomers is relatively higher, producing a number of non-selective binding sites and causing non-specifical interactions between the imprinted nanospheres and template molecules, which can result in poor imprinting performance.

Adsorption Capacity (mg g-1)

Fig. 6. SEM images of nanospheres prepared at different volume ratios of oil to water phase. (A) 1:13.3, (B) 1:20, (C) 1:30, (D) 1:40 and (E) Size distribution of nanospheres prepared at different volume ratios of oil to water phase.

40 35

NIPs MIPs

30 25 20 15 10 5 0 1:1:20 1:2:20 1:4:20 Molar ratio of CAP to MAA to EDMA

Fig. 7. Effects of the molar ratio of CAP to MAA on imprinting performance.

3.6. Morphological characterization of imprinted nanospheres The SEM images and the size distribution of MIPs and NIPs nanospheres obtained in the above optimal conditions are shown in Fig. 8. These results indicate that obtained nanospheres are relatively uniform spherical morphology, the sizes of MIPs

and NIPs nanospheres are 543.9 nm and 565.7 nm in diameter, respectively. In addition, the physical properties such as the specific pore volume and specific surface area of MIPs and NIPs were measured

100

100

90

90

80

80

70

70

Distribution (%)

Distribution (%)

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60 50 40 30

60 50 40 30

20

20

10

10

0

10 100 Particle diameter (nm)

1000

93

0

10 100 Particle diameter (nm)

1000

Fig. 8. SEM images and size distributions of MIPs and NIPs nanospheres. (A) SEM image of MIPs nanospheres; (B) SEM image of MIPs nanospheres; (C) Size distribution of MIPs nanospheres, span is 0.125; (D) Size distribution of NIPs nanospheres, span is 0.08.

NIPs MIPs

20 15 10 5 0 CAP

EM Antibiotics

FF

Adsorption capacity (mg g-1)

Adsorption capacity (mg g-1)

25

25 20 MIPs NIPs

15 10 5 0 0

50

100 150 200 Time (min)

250

300

Fig. 9. Selectivity of the imprinted nanospheres for CAP.

Fig. 10. Adsorption kinetics curve of MIPs and NIPs nanospheres.

by the nitrogen sorption porosimetry. The results indicate that the pore volume and surface area of the MIPs are respectively 0.221 cm g  1 and 168.660 m2 g  1, they are significantly larger than these of NIPs. During the preparation of MIPs, a lot of threedimensional cavities were produced in the imprinted nanospheres because of the presence of template molecules. After the template molecules were washed, it led to the greater pore volume of the imprinted nanospheres than that of the nonimprinted nanospheres. Furthermore, the specific surface area also obtained the same conclusion.

UV–vis spetrophotometry. The concentration of the substrate is 50 mg mL  1 methanol–water (5:95, v:v) solution. The results are shown in Fig. 9. It can be seen that adsorption capacity of the imprinted nanospheres for CAP is much more than that of FF and EM, which further demonstrates the imprinted microspheres have good selective recognition for the template molecule CAP.

3.7. Selectivity of the imprinted nanospheres To further study the adsorption selectivity of the imprinted nanospheres, the adsorption capacities of the imprinted nanospheres for three substrates (EM, FF and CAP) were determined by

3.8. Absorption kinetics of the imprinted nanospheres for CAP The adsorption dynamic curves of MIPs and NIPs are shown in Fig. 10. It can be seen that the adsorption capacity of CAP binding to the MIPs is much higher than that of the NIPs. Moreover, for the MIPs, there exists a rapid dynamic adsorption of CAP to the MIPs. In the first 60 min, the adsorption capacity increases fast with increasing adsorption time, and then the adsorption capacity keeps a constant in the afterward time. However, there is no large

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140

120 100

MIPs NIPs

80 60 40 20

Adsorption capacity (mg g-1)

Adsorption capacity (mg g-1)

140

120 100 80 60 40 20 0

0 0

1000 2000 3000 4000 5000 6000 Concentration of CAP (µg

0

mL-1)

1000 2000 3000 4000 5000 6000 Concentration of CAP (µg mL-1)

Fig. 11. (A) Static equilibrium adsorption isotherm of CAP on MIPs and NIPs nanospheres; (B) Plotted curves for adsorption isotherm of MIPs and NIPs.

difference among the adsorption capacity of the NIPs for CAP with the time. 3.9. Adsorption isotherm of the imprinted nanospheres It is significant to investigate adsorption isotherm of the imprinted nanospheres for further study. The methodology usually used to investigate the thermodynamic adsorption properties of MIPs is to obtain a plotted adsorption curve. We carried out the static equilibrium adsorption experiments in different CAP concentrations (20–6000 mg mL  1) in methanol–water (5:95, v:v), in which 30 mg nanospheres were mixed with 6 mL CAP solution for 2 h. It can be seen from Fig. 11(A), the adsorption capacity of MIPs and NIPs to CAP are increased gradually with increasing concentration of CAP. Furthermore, the adsorption capacity of MIPs is significantly greater than that of NIPs at the equal initial concentration of CAP. It indicates that the imprinted nanospheres have good imprinting effect to CAP. The reason is that there are apparent differences in tri-dimensional structure between the MIPs and the NIPs. In the MIPs, there are lots of sites and cavities that are complementary to CAP in size and shape, and they are contributive to high effective selectivity for CAP. But, to NIPs, there are no sites and cavities complementary to the template and so their selectivity for CAP is worse. In addition, the plotted adsorption isotherm of MIPs is shown in Fig. 11(B). The plotted curve is approximated by modified Langmuir isotherm Eq. (2) with parameters a¼158.78, b¼0.01564 and c¼ 0.3867, and the correlative coefficient R2 is equal to 0.9775. The results indicate that the maximum theoretical absorption capacity of MIPs is up to 158.78 mg g  1 y¼

abx1c 1 þbx1c

ð2Þ

4. Conclusions This study demonstrates the feasibility of synthesizing relatively uniform MIPs nanospheres by premix membrane emulsification technique. Molecularly imprinted nanospheres for CAP were prepared, in which MAA was used as template molecules and EDMA was as cross-linkers. The optimal conditions were as follows: 1.4 mm of the membrane pore size, 2.00 MPa of transmembrane pressure, 5 times of transmembrane number, 1:20 of the volume ratios of oil–water, 1:2:20 of the molar ratio of CAP–MAA to EDMA. The resulted nanospheres are relatively uniform with a diameter from 300 nm to 800 nm. Furthermore, the selective adsorption experiment indicates that the imprinted nanospheres have good selectivity

for CAP. Additionally, adsorption kinetics and adsorption isotherm of the imprinted microsphere show that the adsorption equilibrium time is around 60 min and the maximum theoretical static binding capacity is up to 158.78 mg g  1.

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