Elsevier

Biomaterials

Volume 29, Issue 13, May 2008, Pages 2138-2142
Biomaterials

Imprinting effect of protein-imprinted polymers composed of chitosan and polyacrylamide: A re-examination

https://doi.org/10.1016/j.biomaterials.2008.01.019Get rights and content

Abstract

In this paper the imprinting effects recently reported with protein-imprinted polymers based on chitosan and polyacrylamide are re-assessed. Molecularly imprinted polymers (MIPs) from which the embedded template molecules were removed by washing with a solution of sodium dodecyl sulfate and acetic acid were prepared. In batch template rebinding experiments, the MIPs displayed quite high template binding capacity as reported previously. However, the non-imprinted polymers (NIPs), after washing with the same solution, also showed large binding capacity nearly equal to that of the MIPs. X-ray diffraction and scanning electronic microscope investigations confirmed remarkable property changes of the NIPs after the washing process. These findings indicate that the non-specific adsorption resulting from the template removing process rather than the imprinted sites generated on the MIPs themselves may account for the high template rebinding capacity of the reported protein-imprinted polymers.

Introduction

Molecular imprinting is an inexpensive method for the synthesis of tailor-made recognition materials by copolymerizing suitable functional monomers in the presence of desired template molecules. Compared with natural recognition materials such as antibodies, molecularly imprinted polymers (MIPs) offer advantages like stability, specific recognition and ease of mass preparation, and thus have been widely applied in a number of areas including chromatographic applications, separation, solid phase extraction, catalysis and sensors [1], [2]. To date, the imprinting of small molecules has been well-established and considered almost routine. However, the imprinting of biomacromolecules such as proteins continues to be a significant challenge due to the difficulties with large molecular sizes, structural complexity, environmental sensitivity of the templates, and significantly reduced non-covalent template–monomer interactions in aqueous media [3], [4], [5]. Despite these difficulties, there is still a strong incentive to synthesis MIPs for the use of biosensors, diagnostics, bioseparation, etc, and quite a few attempts have been made with more or less success. The progress in this field has been comprehensively reviewed [6], [7], [8], [9].

Most of the protein-imprinted polymers demonstrated to date show very low template rebinding. Few exhibit rebinding capacity high enough for some applications such as bioseparation. Among these, Yang and co-workers [4] produced the protein-imprinted nanowires based on cross-linked polyacrylamide (PAM) with a large rebinding capacity of up to 30 mg/g nanowires. This can be ascribed to the surface-situated imprints and large surface area of the nanowires. Guo's group [10], [11], [12] fabricated bovine hemoglobin (Hb)-imprinted polymers by the combined use of chitosan, a natural polymer abundant of –NH2 and –OH groups, and cross-linked PAM. The MIPs were prepared by physical entrapment [10], chemical grafting [11] of soft PAM gels into porous cross-linked chitosan beads or by forming homogenous semi-interpenetrating polymer network (s-IPN) hydrogel monolith based on PAM and chitosan [12]. These MIPs presented a high Hb rebinding capacity of above 20 mg/g wet gels, while the NIPs bound very little Hb. Following their work, we recently reported on bovine serum albumin (BSA)-imprinted hydrogels by homogenous graft copolymerization of acrylamide on chitosan, and the MIPs exhibited even higher template rebinding and better stability than those formed from the PAM/chitosan s-INP [13]. However, now we have found that these high binding results are difficult to explain reasonably, apart from the –NH2 and –OH groups in the macromolecular chains of chitosan which may interact with protein template molecules through relatively strong hydrogen bonding. Moreover, we have noticed from Refs. [12], [13] that, in the rebinding tests, the total template amount bound onto the MIPs was even larger than that used for generating imprints during MIP synthesis. This is not in agreement with the general fact that the former amount accounts for only a small fraction of the latter when non-covalent imprinting method is adopted, since the yield in binding sites relative to the amount of imprint molecule used is low [1]. The high rebinding capacity of the MIPs might be attributed to the significant non-specific binding not due to the imprinted sites. However, this is not consistent with the very low template binding observed on the NIPs. Therefore, we deduce that there may be some problem associated with some recent studies including those of Guo and ourselves.

The purpose of this paper is to ascertain the underlying problem and examine the actual imprinting effect of the protein-imprinted polymers based on chitosan and PAM. We re-prepared these MIPs and the corresponding NIPs. The template-containing MIPs were washed with a solution of sodium dodecyl sulfate (SDS) and AcOH to remove the embedded template molecules and a half of the NIPs were also washed in the same way. Batch template rebinding experiments were carried out to compare the protein template binding capacity of these media. Scanning electronic microscope (SEM) and X-ray diffraction (XRD) were, respectively, used to investigate the morphology and structure of these polymer matrixes.

Section snippets

Materials

Two kinds of chitosan powder were used: one was purchased from Yuhuan Biochemical Co., Ltd. (Zhejiang, China) with viscosity average MW of about 50,000 (denoted by Chitosan 5); and the other from Boao Biotechnology Co. (Shanghai, China) with viscosity average MW of about 500,000 (Chitosan 50). The deacetylation degree of both chitosan samples was about 90%. Acrylamide (AAm) and N,N′-methylenebisacrylamide (MBA) of electrophoresis grade were purchased from Dingguo Biotech Co., Ltd. (Beijing,

Protein template rebinding

In the published work of Guo and co-workers [12] and our group [13], both Hb-imprinted and BSA-imprinted hydrogels were obtained through granulating the resultant monolithic hydrogel, which was formed from solution polymerization in the presence of the imprint molecules. Obviously, the template amounts embedded in unit weight of the MIP hydrogels before the template removal step can be easily estimated according to the given synthesis recipes, being about 12 and 14 mg/g wet gels, respectively.

Conclusions

Both Guo's group [10], [11], [12] and our group [13] recently reported on the protein-imprinted polymers composed of chitosan and PAM, which showed successful template imprinting effect with quite high template rebinding capacity. However, such high template rebinding cannot be reasonably explained. In this study, we have found that the corresponding NIPs, after washing with the AcOH–SDS solution in the same way as the MIPs for the protein template removal, achieved high template binding

Acknowledgement

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 20574038).

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