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Combinatorial synthesis and screening of uniform molecularly imprinted microspheres for chloramphenicol using microfluidic device.


Molecularly imprinted polymers (MIPs) have been demonstrated possessing unique and predetermined selectivity for target molecules and have been developed for a variety of applications including chromatography, enzymatic catalysis, solid-phase extraction, and sensor technology (1-9).

However, the most widely used method for preparing MIPs was by bulk polymerization, followed by grinding into particles. These particles are polydisperse, irregular and usually contain a large portion of waste fine particulate material. Moreover, additional sieving is required to obtain a more narrow size distribution and to remove fine particles, which makes the method tedious and time-consuming (10). In recent years, because spherical MIP particles have numerous advantages in terms of the process and performance in their final applications, some approaches for preparing MIP microspheres have been recently reported (11), (12), such as suspension polymerization in liquid perfluorocarbon and mineral oil (13), (14), multi-step swelling (15), (16) and grafting polymerization directly to a suitable support (17), (18). In addition, Zourob et al. first reported the preparation of uniform MIP microspheres in a spiral-shaped microchannel (19). In this method, MIP microspheres are formed in one-step continuous flow process, but each time it only can be performed under one condition (or formulation) to produce droplets and microspheres.

On the other hand, ideal MIPs network contains synthetic receptors that are complementary in size, shape and functional group orientation to the template molecule. Both the morphology of pore in the polymer and the chemical microenvironment of binding site are critical to the overall performance. So preparation of the optimal MIPs usually involves time-consuming trial and error, which includes selection of different functional monomers, ratio of template to functional monomers and temperature of polymerization, et al. Therefore, imprinted polymers are ideal candidates for combinatorial synthesis and its screening technologies. Recently, combinatorial methods have been used to develop highly selective MIPs (20). In 2007, M. T. Koesdjojo et al. described a semiautomated procedure for the synthesis and screening of a large group of molecularly imprinted hulk polymers (21).

In this article, a novel method for combinatorial synthesis and screening of MIP microspheres using microfluidic device is reported, in which chloramphenicol (CAP) is used as a model molecule. This new microfluidic device containing twelve pairs of "h" shape microchannels was designed to form droplets and MIP microspheres via controlled suspension polymerization. This method combines molecular imprinting and microfluidic device with the combinatorial chemistry approach, allowing rapid screening and optimization of MIPs, moreover, the size of MIP microspheres created is very uniform.



To remove the inhibitor before polymerization, methacrylic acid (MAA) and 4-vinylpyridine (4-VP) was distilled under vaccum, while ethylene glycol dimethacrylate (EDMA) was washed with I M aqueous sodium hydroxide, dried over [MaSO.sub.4], and stored with molecular sieves at 4[degrees]C until required. CAP. florfenicol (FF), and tetracycline (TET), MAA, 4-VP, acrylamide (AA), EDMA, and 2,2-azobisiso-butyronitrile (AIBN) were purchased from Sigma-Aldrich. Poly (vinyl alcohol) was obtained from Fluka. Ethyl acetate and chloroform were obtained from Beijing Plant of Chemicals. All other chemicals are analytical or HPLC grades.

Microfluidic Device

Figure 1 shows the schematogram of microfluidic device. The microfluidic device made of glass is fabricated in our laboratory, and it contains twelve pairs of identical "h" shape microchannels, and consists of a bottom and cover plate. Each pair of "h" shape microchannel in the bottom plate of 1.0-mm thickness has three reservoirs and three microchannels, while the cover plate (1-mm thickness) has 0.5-mm diameter drilled holes to facilitate access to the microchannels. After the surface of the bottom plate was mediated with 1.5% (w/v) hydrofluoric acid, the cover plate was bonded to the bottom plate keeping a high pressure of 1.5 atm. at 25[degrees]C for 24 h. In addition, in each "h" shape microchannel, microchannel 1 is about 10-[mu]m width, microchannel 2 and 3 are about 150-[mu]m width, and the depth of all microchannels is about 45 [micro]m.

Preparation of MIP Microspheres and Control Polymer

Uniform-size droplets and spherical microspheres are prepared via controlled suspension polymerization in a "h"-shaped microchannel using water [with 1.5% polyvinyl alcohol (PVA)] as continuous phase, the schematic diagram is shown in Fig. 2, in which 12 pairs of "h"-shaped microchannels are used simultaneously to produce droplets under individual condition. Different functional monomers (MAA, 4-VP, AA), template molecule (CAP), crosslinker (EDMA), initiator (AIBN) were dissolved in porogenic solvents ethyl acetate--chloroform (4:1. v/v). The components were mixed and purged with nitrogen for 10 min. This pre-polymerization mixture is used as the oil phase and pumped into water continuous phase by a multi-channel syringe pump. Droplets were formed when the oil phase met the water in the junction. The resulted droplets within the microfluidic device are introduced to the glass vials containing water with 1.5% PVA, polymerization was performed at 4[degrees]C under magnetic stirring, and exposed to four ultraviolet (UV) light (60-80 mw [cm.sup.-2]) at a peak wavelength of 365 nm for 24 h to rapid initiate polymerization reaction. Corresponding control polymer microspheres (non-molecularly imprinted polymer. NIP) were also synthesized following the exactly same procedure but excluding the template CAP from the formulation.

To remove the template CAP and PVA, the microspheres are collected, filtered, and washed with distilled water. ethanol, ethyl acetate/acetic acid (9:1, v/v), ethanol. and distilled water, respectively. And the microspheres are finally dried in a vacuum overnight.

Characterization of Microspheres by Scanning Electron Microscope

For scanning electron microscope (SEM) observation, the obtained microspheres are dried at 60[degrees]C and then attached to silver papers and coated with a gold layer. A scanning electron microscope (JSM-6700, JEOL SEMI Japan) is used for the morphology observation.

Screening and Evaluation of MIP Microspheres

Evaluation and screening of MIP/NIP microspheres are performed by following binding experiments. Microspheres (6 mg) are mixed with 5 mL of ethanol-water (1:4, v/v) of CAP (120 mg/L), and incubated overnight at room temperature. The solution was then centrifuged at 6000g for 10 min, and supernatant from each vial was collected and detected by ultraviolet-visible (UV-Vis) spectrophotometric analysis.

Calculation of Imprinting Factor

The binding capacity (Q) is defined as mg of substrate bound per 1 g microspheres (MIP or NIP), and calculated by the change of CAP concentration after and before adsorption by Eq. 1, in which [C.sub.0] (mg [mL.sup.-1]) and C (mg [mL.sup.-1]) are the initial concentration and free concentration of substrate in the supernatant, respectively. V (mL) is the volume of adsorption solution. W (g) is the mass of the microspheres.

Q = ([C.sub.0] - C) x V/W (1)

Imprint factor [beta]= [Q.sub.MIP]/[Q.sub.NIP] and [Q.sub.NIP] are the binding capacity of CAP on imprinted and non-imprinted polymer, respectively.

Selectivity of the Imprinted Microspheres

To further verify selectivity of the imprinted microspheres, the adsorption amount of three antibiotics (CAP, FF, and TET) bound on the imprinted microspheres are assessed by static absorption binding experiments, respectively. Figure 3 shows structures of the three antibiotics.

The MIPs and NIPs (6 mg) are placed into six centrifuge tubes of 10 mL, respectively, and added separately 5 mL of ethanol-water (1:4, v/v) solution containing certain concentration of CAP or different analogues, then stirring at 150 rpm overnight. After these samples are centrifuged at 6000 rpm for 10 min, the free CAP, FF and TET in supernatant are measured separately by UV-vis spectrophotometry at 276, 224, and 355 nm.


We describe an approach for fast screening and synthesis of uniform MIP microspheres within a microfluidic device. To obtain uniform-size MIP microspheres of CAP under a variety of conditions, a series of experiments were designed and carried out. The pre-polymerization mixture compositions used to synthesize the MIP microspheres are shown in Table 1, in which three functional monomers (MAA, AA, 4-VP) are employed to investigate and produce MIP microspheres.

TABLE 1. Pre-polymerization mixture formulation used to screen
MIP microspheres of CAP

Run  MIP/NIP     CAP     MAA  Acrylamide    4-VP    EDMA    AIBN
No.           (mmol)  (mmol)      (mmol)  (mmol)  (mmol)  (mmol)

1.   MIP         0.5     0.5           -       -      12    0.20

     NIP           -     0.5           -       -      12    0.20

2.   MIP         0.5     0.1           -       -      12    0.20

     NIP           -     0.1           -       -      12    0.20

3.   VHP         0.5     1.5           -       -      12    0.20

     NIP           -     1.5           -       -      12    0.20

4.   MIP         0.5     2.0           -       -      12    0.20

     NIP           -     2.0           -       -      12    0.20

5.   MIP         0.5     2.5           -       -      12    0.20

     NIP           -     2.5           -       -      12    0.20

6.   MIP         0.5     3.0           -       -      12    0.20

     NIP           -     3.0           -       -      12    0.20

7.   MIP         0.5       -         0.5       -      12    0.20

     NIP           -       -         0.5       -      12    0.20

8.   MIP         0.5       -         0.1       -      12    0.20

     NIP           -       -         0.1       -      12    0.20

9.   MIP         0.5       -         1.5       -      12    0.20

     NIP           -       -         1.5       -      12    0.20

10.  MIP         0.5       -         2.0       -      12    0.20

     NIP           -       -         2.0       -      12    0.20

11.  MIP         0.5       -         2.5       -      12    0.20

     NIP           -       -         2.5       -      12    0.20

12.  MIP         0.5       -         3.0       -      12    0.20

     NIP           -       -         3.0       -      12    0.20

13.  MIP         0.5       -           -     0.5      12    0.20

     NIP           -       -           -     0.5      12    0.20

14.  MIP         0.5       -           /     0.1      12    0.20

     NIP           -       -           -     0.1      12    0.20

15.  MIP         0.5       -           -     1.5      12    0.20

     NIP           -       -           -     1.5      12    0.20

16.  MIP         0.5       -           -     2.0      12    0.20

     NIP           -       -           -     2.0      12    0.20

17.  MIP         0.5       -           -     2.5      12    0.20

     NIP           -       -           -     2.5      12    0.20

18.  MIP         0.5       -           -     3.0      12    0.20

     NIP           -       -           -     3.0      12    0.20

Each kind of resulted MIP microspheres was evaluated by the binding amount for CAP. Each kind of MIP microspheres (6 mg) was added into 5 mL of ethanol-water (1:4, v/v) solution containing CAP. After the mixture is incubated overnight at room temperature, supernatant are collected and analyzed by UV-Vis spectrophotometric analysis to quantify the free CAP in solution. The amounts of CAP hound on the MIPs are obtained by subtracting the free CAP in solution from the initial amount. Then the imprinting performance was evaluated by imprinting factor ([beta]).

Figure 4 show's the imprinting factor of each kind of MIP microspheres. In Fig. 4. MIP and NIP microspheres 1-6 were synthesized using the acidic monomer MAA. MIP and NIP microspheres 7-12 were synthesized using the neutral monomer AA. MIP and NIP microspheres 13-18 were synthesized with the basic monomer 4-VP. As illustrated in Fig. 4. the MIP microspheres obtained with MAA possess better imprinting performance, and the highest imprinting factor is obtained by M1P microsphere No. 5. Therefore, from the three monomers selected initially, MAA is proved to be the most suitable functional monomer for the imprinting of CAP. and polymerization formulation No. 5 is demonstrated to be the most optimal condition for the synthesis of CAP. The probable reason is as follows: in the structure of the template molecule CAP, there are HO--,--[NO.sub.2], and--NH--functional groups. MAA is the best choice of monomer since it not only can form electrostatic interaction with the basic functional group of CAP but also interact with CAP by hydrogen bond. Instead, AA mainly interacts with CAP by hydrogen bond, and 4-VP produce interaction with CAP through hydrogen bond or van der waals force. So, MAA can cause stronger interaction than AA and 4-VP with CAP.

Under the condition of polymerization formulation No. 5, MIP microspheres of CAP were produced with the microfluidic device, and the resulted MIP microspheres were characterized by scanning electron microscope. The result is shown in Fig. 5, which suggests that the obtained microspheres have good monodispersity. The MIP microspheres have low coefficient of variation values <5%, and the average particle size is about 29 pm. In addition, the droplets produced within the microchannel were observed by optical microscope. The photo of droplets obtained by optical microscope is shown in Fig. 6. It reveals the droplets are also very uniform.

In addition, we select three kinds of antibiotics including CAP, FF, and TET to study the selectivity of the imprinted microspheres of CAP, in which their concentrations are 120 mg [L.sup.-1] in ethanol-water (1:4, v/v), and the results are shown in Fig. 7. It can be seen from Fig. 7, the imprinted microspheres possess good selectivity to CAP. The imprinted microspheres have much higher adsorption amount for CAP than that of other two antibiotics. In addition, MIPs have much higher adsorption amount for CAP than NIPs. The reason is that there are apparent differences in tri-dimensional structure between the MIPs and the NIPs. In the MIPs, there are a lot of sites and cavities which are complementary to CAP in size and shape. and they are contributive to high selectivity for CAP. To NIPs, however, there are no sites and cavities complementary to the template and so their selectivity for CAP is worse.


To the best of our knowledge, it is the first time that a novel microfluidic device is designed to screen and synthesize uniform MIP microspheres of CAP, in which the new microfluidic device containing 12 pairs of "h" shape microchannels was utilized to produce MIP microspheres via controlled suspension polymerization. MIP microspheres of CAP were prepared by a combinatorial approach using water with 1.5% PVA as continuous phase, and ethyl acetate-chloroform (4:1, v/v) as porogenic solvents, and MAA, 4-vinylpyridine. AA as functional monomers. The results demonstrate that the imprinted microspheres obtained with MAA as a functional monomer have the best imprinting effects.

In the microlluidic device, 12 kinds of imprinting conditions can be performed simultaneously, so this combinatorial protocol is well suited for fast and efficient screening and optimization of synthesis for uniform-size MIP microspheres. Moreover, here we only describe a method to screen and synthesize uniform-size MIP microspheres. In fact, it is possible to design more than twelve pairs of "h" shape microchannels to screen and synthesize not only MIP microspheres but other microsphere materials with good monodispersity. Thus, the combinatorial protocol will be more rapid and efficient to run a high-throughout screening and optimization for the synthesis of microspheres. So this technique is very useful and promising.


AA    Acrylamide
AIBN  2,2-Azobisiso-butyronitrile
CAP   Cholramphenicol
EDMA  Ethylene glycol dimethacrylate
FF    Florfenicol
MAA   Methacrylic acid
MIP   Molecularly imprinted polymer
NIP   Non-molecularly imprinted polymer
PVA   Polyvinyl alcohol
TET   Tetracycline
UV    Ultriviolet


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Correspondence to: Jiandu D. Lei; e-mail:

Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 20976179; Contract, grant sponsor: Beijing Natural Science Foundation; contract grant number: 2092027.

Published online in Wiley Online Library (

[c] 2012 Society of Plastics Engineers

Xingyong Liu, (1) Jiandu Lei (2)

(1.) School of Materials and Chemical Engineering, Sichuan University of Science and Engineering, Zigong 643000, People's Republic of China

(2.) State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People's Republic of China

DOI 10.1002/pen.23159
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Author:Liu, Xingyong; Lei, Jiandu
Publication:Polymer Engineering and Science
Article Type:Report
Geographic Code:9CHIN
Date:Oct 1, 2012
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