научная статья по теме RECOGNITION OF 6-BENZYLADENINE USING MOLECULARLY IMPRINTED MEMBRANE ON CELLULOSE ACETATE SUPPORT Химия

Текст научной статьи на тему «RECOGNITION OF 6-BENZYLADENINE USING MOLECULARLY IMPRINTED MEMBRANE ON CELLULOSE ACETATE SUPPORT»

ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2008, том 63, № 10, с. 1097-1102

^=ОРИГИНАЛЬНЫЕ СТАТЬИ =

УДК 543

RECOGNITION OF 6-BENZYLADENINE USING MOLECULARLY IMPRINTED MEMBRANE ON CELLULOSE ACETATE SUPPORT1

© 2008 г. X. J. Qu*, Q. X. Meng*, S. Y. Ai*, J. Zhou*, L. S. Zhu**

* College of Chemistry and Material Science, Shandong Agricultural University Taian 271018, P. R. China **College of Resources and Environment, Shandong Agricultural University Taian 271018, P. R. China Received 01.12.2006, in final form 11.03.2008 r.

A molecularly imprinted polymer membrane was prepared on cellulose acetate support by photopolymerization of methacrylic acid and a cross-linker, ethyleneglycol dimethacrylate, in the presence of the template molecules of 6-benzyladenine (BA). The polymeric membrane morphologies were visualized by scanning electron microscopy and its selectivity was evaluated by permeation test. Association ratio and apparent association constant of the complex formed between methacrylic acid and BA were determined by cyclic voltammetry, and are 1 : 1 and 204.9, respectively. These results indicated that there existed some complementary cavities on the imprinted membrane corresponding in size, shape, and functional groups to the template molecules of BA. Hence, the imprinted membrane was able to recognize BA. It is predicted that this molecularly imprinted membrane may be applicable to the assay of BA or for the preparation of a molecularly imprinted polymer sensor for the determination of BA in plant samples.

During the last few years, considerable progress has been made in the field of molecular imprinting. Owing to the current fascination with nano-technology and "designer" materials, much attention in the technique has gained [1-5]. Molecular imprinting is a process that employs a target molecule as the template and makes selective recognition sites in synthetic polymers, in which a matrix was polymerized around the template molecules, followed by removal of the template. This leaves host cavities that, ideally, retain a shape and functional group complementarity to the guest-template. Recently, molecularly imprinted membranes have become a novel promising tool of analytical chemistry. The transport of metal ions or organic molecules across the membranes has been studied as a means of separation, concentration and recognition [6-11].

Cytokinin (CK) compounds [12-15] that function as plant hormones are adenine molecules connected to an N6 side chain. Metabolic pathways produce various structural modifications to the adenine moiety and/or the side chain that affect cytokinin function, activity, stability, and transport in plant. Cytokinins take part in the control of cell division, chloroplast development, bud differentiation, shoot initiation and growth or leaf senescence. In contrast to the wide knowledge of CK effects, the mechanisms of CK action remain largely unknown [16]. Since molecular imprinted polymers can act as biomimetic receptor to recognize specific molecules, the studies on molecularly imprinted polymers (MIPs) of cytokinins have a significant

1 Supported by the National Natural Science Foundation of China (No. 20477022).

meaning for the exploration of the action mechanisms and the high selective determination of cytokinins.

BA is one of cytokinins. Furthermore, of all cytoki-nins, BA is most widely used for plant tissue culture, which is an important part of modern biotechnology. In the present work, molecularly imprinted polymer membranes (MIPM) containing artificial recognition sites for BA have been prepared by photopolymerization using BA as template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker, and N,N-dimethylformamide (DMF) as a porogen. The association constant of the template molecules BA and the functional monomer was determined. The surface patterns of the imprinted membrane was characterized by scanning electron microscope (SEM). The selectivity of the imprinted membrane was evaluated by adsorption and permeation studies for BA and its derivative, 6-(furfurylamino)purine (KT), which has a similar structure. Based on the experiment results, the adsorption and permeation mechanisms of BA and KT were discussed, the results of studies provided experimental basis for the study of mechanisms of CK action in plants.

EXPERIMENTAL

Reagents and materials. MAA and EGDMA were obtained from J&K Chemical Ltd (Beijing, China) and were distilled in vacuum prior to use in order to remove stabilizers. 2,2'-Azobis (2-isobutyronitrile) (AIBN) was supplied by Special Chemical Reagent Factory of Nankai

N^NH

N NH

HN

/ \

N ___O HN

n=/ Г

N

N

Reagent Membrane A Membrane Q Membrane R

BA* 2.7 0.6 0.6

EGDMA 93.1 46.6 23.3

AIBN 0.2 0.4 0.4

MAA 4.0 52.4 75.7

t

MP-2 polarogrphic analyzer/stripping voltammeter

6-Benziladenine (BA) 6-(Furfurylamino)purine (KT)

Fig. 1. Structures of 6-Benzyladenine and 6-(Furfurylami-

no)purine.

University (Tianjin, China) and used without further purification. BA and KT, illustrated in Fig. 1, were purchased from Acros Organics (Geel, Belgium). Cellulose acetate membrane filter (pore size, 0.45 ^m; for porous solid support in the polymer matrix) was purchased from Whatman (Middlesex, UK). Buffer solutions of pH 3.8 was prepared with acetic acid (HOAc)/sodium acetate (NaOAc)ac-cording to ref. 17. All reagents are of analytical grade except for AIBN, which is of chemical purity grade. Pure nitrogen gas (99.99%) was used for deaeration. Doubly distilled water was used throughout the experiment.

Hitachi model S-570 scanning electron microscope (SEM) and a MP-2 polarographic analyzer/stripping voltammeter (Shandong No.7 Telecommunicational Equipment Factory, China) were used.

Preparation of molecularly imprinted polymer membranes. MIPM were prepared with BA as a template, MAA as a functional monomer and EGDMA as a cross-linker. The molar ratio of the functional monomer to the template was chosed to be 4 : 1 [18-20]. In order to obtain thin, flexible, and mechanically stable membranes, MIP and nonimprinted polymer were polymerized onto a cellulose acetate filter membranes (pore size, 0.45). The typical preparation of the molecularly imprinted polymer membrane was carried out as follows. A 0.2253 g (1 mmol) amount of BA was mixed with 0.3444 g (4 mmol) of MAA, 7.9288 g (40 mmol) of EGDMA, 0.0200 g of AIBN and 8 mL of the solvent (DMF). The mixture was purged with nitrogen for 5 min and then outgassed from nitrogen with ultrasonic wave for 10 min. Subsequently, cellulose acetate membrane filter (CAMF) was immersed

Table 1. The composition of the membrane in wt % of the total components in the original mixture

counter electrode DME -

Ag|AgCl -membrane

d

* BA was dissolved in 8 mL of N,N-Dimethylformamide in each case.

Fig. 2. Schematic diagram of the experimental set-up for selectivity evaluation.

in the mixture for 20 min. The membrane was then sandwiched in between two quartz slides. To complete polymerization, the slides with the membrane were exposed to UV radiation (365 nm, intensity 20 w m-3) for 6 h at 4°C. After polymerization, the template was extracted with DMF three times and then with 0.1 M acetate buffer (pH 3.8) until the template molecule could no longer be detected by a single-sweep polarography. The composition of the membrane in wt% of the total components in the original mixture is shown in Table 1 (A). Other membranes with different composition (Table 1, Q and R) were also prepared.

The non-molecularly imprinted polymer membrane (NIPM) for control experiments were prepared in the same manner as membrane A except that the monomer mixture did not contain BA.

Membrane responses. The adsorption and permeation of the prepared MIPM were monitored in order to evaluate their selectivity to BA. Transport studies were carried out in a H-shaped cell consisting of two detachable parts (Fig. 2). The membrane, with an exposed cross-sectional area of 7.069 cm2, was placed between the two halves of the cell. The halves were held together with a screw-actuated clamp that compresses an o-ring seal to tightly secure the connection. Acetate buffer (0.1 M, pH 3.8) was used as a background electrolyte, and it was used as a receiving phase filling in one side of the membrane. Another side of the membrane was a solution containing 0.5 mM BA or KT in the background electrolyte as a source solution. After regular time, a portion of the receiving solution was analysed by single-sweep polarogra-phy to determine the amount of CK that was transported across the membrane. Program control and data processing are conducted automatically by the microcomputer system. The three-electrode system used in this study contained a dropping mercury working electrode (DME), a platinum wire counter electrode and a Ag | AgCl reference

electrode. Solutions were deoxidized by bubbling purified nitrogen for at least 10 min prior to the measurements.

RESULTS AND DISCUSSION

Interactions between BA and MAA . Covalent interac -tions between a template and complementary functional monomers have been extensively used in the technology of molecular imprinting [21]. According to this approach, a polymerizable derivative of the analyte is synthesized and used as a template. The subsequent extraction of such a polymerizable template requires cleavage of some covalent bonds. Because of the cleavage reducing the numbers of potential templates, we focused on another approach that employs non-covalent interactions between a tem plate and functional monomers. In addition to good versatility, this approach provides fast and reversible binding of the template. In this work, methacrylic acid was chosen as a functional monomer as it is capable of showing high se -lectivity and leads to polymers that retain the template more strongly [21].

Solvent (referred to as porogen) plays an important role in a molecular imprinting process [22-24]. The h

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