QCM–FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III

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Abstract

In this work, we describe a method of constructing a film of linear poly(glycidyl methacrylate) (PGMA) polymer onto the surface of quartz crystal microbalance (QCM) electrode as a coating material that allows easy coupling of heparin molecules onto the electrode and facilitates the determination of the interaction between heparin and antithrombin III (AT III). The PGMA film was characterized with atomic force microscopy (AFM) and infra-red spectroscopy. The coupling of heparin was accomplished in one step solution reaction. A home-made quartz crystal microbalance–flow injection analysis (QCM–FIA) system with data analysis software developed in our laboratory was used to determine the interaction. The interactions between immobilized heparin and AT III were studied with various concentrations under various conditions. The obtained constants are kass = (1.49 ± 0.12) × 103 mol−1 l s−1, kdiss = (3.94 ± 0.63) × 10−2 s−1, KA = (3.82 ± 0.33) × 104 mol−1 l.

Introduction

As an extremely sensitive surface mass sensor, quartz crystal microbalance (QCM) has been used for the measurement of mass change in a variety biological studies such as DNA hybridization in solution phase (Ebersole et al., 1990, Caruso et al., 1997), piezoimmuoassay (Park et al., 2000, Fung and Wong, 2001), proteins adsorption on solid surface (Höök et al., 2002, Tanaka et al., 2001), and studies related to cell–substrate interactions in situ and measurement of the dynamics of exocytosis and vesicle retrieval at cell populations (Wegener et al., 2001, Cans et al., 2001). Combined with flow injection analysis (FIA), QCM allows on-line monitoring of the analyte binding, and thus provides a much more convenient tool for the determination of analytes or real-time study of analyte interactions. QCM–FIA has been applied to piezoimmuoassay by Navrátilová et al. (2001). Liu et al., 2003a, Liu et al., 2003b performed kinetic analysis of the interactions between proteins and small molecular agents with QCM–FIA. They calculated the kinetics constants, and compared the difference in affinity for various proteins and small molecular agents. Lau et al. (2000) carried out the determination of fructose and analysis of kinetics constants by using the affinity mass sensor based on QCM–FIA. Decker et al. (2000) isolated a human pancreatic secretory trypsin inhibitor mutant from a large phagemid library using QCM–FIA.

The immobilization of ligand molecules is a crucial procedure for the QCM measurement. It is necessary that ligand molecules be fixed onto the surface of electrode easily under relatively mild conditions. The obtained electrode coatings should be chemically stable during the measurement and retain the biological activity for target samples. Moreover, the electrode coatings must be uniform, rigid, and as thin as possible (Babacan et al., 2000, Luong and Guibault, 1991).

The method of self-assembled monolayer (SAM) is commonly used to immobilize ligand molecule (Fung and Wong, 2001, Liu et al., 2003a, Liu et al., 2003bSi et al., 2002, Su and Li, 2001, Storri et al., 1998, Katz, 1990). Generally, a self-assembled functional monolayer is first formed on the surface of electrode as a base coat through self-assembly of mercapto-compound onto the surface of gold, then a layer of ligand molecules can be constructed on the base coat through a cross-linker (such as glutaraldehyde) or a direct covalent bonding after a chemical activating procedure. Heterobifunctional thiolation cross-linker has also been applied to the immobilization of antibody onto the gold surface with SAM method (Park et al., 2000). The base coat can also be formed by physisorption, including direct physisorption of biomolecules (Uttenthaler et al., 1998, Cheng et al., 2002, Davis and Leary, 1989), organic silane agents (Zhou et al., 1997, König and Grätzel, 1994, Eun et al., 2002), and polymers such as polyethylenimine (PEI) (Eun et al., 2002, Bunde et al., 2000), diamine (EDA) (Saber et al., 2002), copolymer of hydroxyethyl methacrylate and methyl methacrylate(Wu et al., 1999), styrene–butadiene–styrene polymer (He et al., 2002), or polyethylene–co-acrylic acid polymer (Chong et al., 2002).

Because the assembly of a SAM is relatively easy and less dependent on special technique and equipment, and the formed SAM is in form of a stable and ordered film (Fung and Wong, 2001), SAM method has been chosen in most of the literatures. However, polymers possess many advantages as base coat. Compared with the costly mercapto-compounds, the monomer materials for the polymers are usually inexpensive and readily available. In addition, the preparation of polymers is simple, and the polymers can be made with many different functional groups to provide great flexibility in coupling different ligand molecules. The polymer thin film on the surface of QCM electrode can be shaped in a simple mode. Therefore, much more attention has recently been focused on the use of polymers as coating material.

Heparin, which belongs to the family of glycosaminoglycan, can interact with many proteins and their interactions regulate many important biological processes. The most significant function of heparin is to prevent the formation of thrombus by means of binding to antithrombin, therefore, heparin has been extensively used in blood transfusion as an anticoagulant. Antithrombin III (AT III) is a member of the serine protease inhibitors superfamily (serpins) (Petitou, 1994), and is the primary anticoagulating factor in blood. It inhibits a number of coagulation proteinases. Normally, AT III is present in plasma in an almost inactive form. When bound to heparin species, it is activated and its antithrombin reactivity toward factor Xa is greatly accelerated (Petitou, 1994). A better understanding of the interaction between heparin and AT III, especially the kinetics information, will benefit the design of new anticoagulants (Desai and Gunnarsson, 1999). Recently, the study of the interaction between heparin and AT III has received considerable interest.

In this paper, the dynamic interaction of heparin with AT III was used to demonstrate the possibility of linear poly(glycidyl methacrylate) (PGMA) as coating material to immobilize heparin molecules onto the surface of QCM electrode. A home-made QCM–FIA system and kinetic analysis software based on genetic algorithm were used in the experiment. The results on the kinetics of the interaction between heparin and AT III will be presented.

Section snippets

Materials and reagents

Linear poly(glycidyl methacrylate) (PGMA) was synthesized in our lab according to the reported procedure (Zhao et al., 1999). Heparin sodium salt (MW 6000–20,000, 140 u mg−1) was purchased from Chang Zhou Institute of Biochemistry (Chang Zhou city, Jiangshu Province, PR China). Antithrombin III (AT III) (MW 60,000) was obtained from Hua Lan Bioengineering Co. Ltd. (Xin Xiang city, Henan province, PR China). All other chemicals were of analytical grade and used as received.

Phosphate buffer

Results and dicussion

Epoxy-activated affinity supports have been widely used in affinity chromatography (AC) (Wheatley and Schmidt, 1999). Epoxy group can easily react with many kinds of functional groups, such as single bondNH2, single bondOH, single bondSH, single bondCOOH, and the reaction can be performed in one step under relatively mild conditions. Therefore, it provides many convenient protocols for us to immobilize different ligand molecules. This method can be used for the immobilization of amino acids, amines, carbohydrates, peptides and proteins

Conclusion

The possibility of using PGMA as a coating material for the sensor electrode for the study of biomolecule interactions was demonstrated using a home-made QCM–FIA system. The results showed that a PGMA polymer film could be easily constructed on the surface of the sensor electrode for further coupling the heparin molecules to detect the interaction of heparin with AT III. The coupling reaction was carried out in one step under relatively mild conditions without other activating procedures. The

Acknowledgements

The work was supported by the NSFC (No. 90408018, 20035010) and Chinese Academy of Sciences (No. KJCX2-SW-H06). We are also grateful to Mr. Liu Gang and Mr. Zhang Ying for their assistance in AFM and FT-IR analysis.

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