Adsorption of 125I-labeled immunoglobulin G, its F(ab′)2 and Fc fragments onto plasma-polymerized films

https://doi.org/10.1016/j.bios.2006.10.021Get rights and content

Abstract

Plasma-polymerized films were formed on flat glass plates using allylamine, acrylic acid, acrolein, and allylcyanide as monomers. Adsorption of 125I-labeled-proteins such as immunoglobulin G (IgG), its F(ab′)2 and Fc fragments, and human serum albumin (HSA) was measured on these plasma-polymerized (PP) films covering the glass plates and on commercially available polymer plates. The adsorption isotherm followed the Langmuir equation, from which the binding constant and amount of saturation binding were estimated. We found that, in general, a cationic surface had higher affinity for protein adsorption than an anionic surface. Among the surfaces examined, the PP-allylamine surface showed the highest binding capacity (264.2 nmol/m2) for F(ab′)2 fragment: it was remarkably high. Of the surfaces examined, the PP-acrylic acid surface showed the lowest binding capacity (12.8 nmol/m2) for F(ab′)2 fragment. The PP-acrylic acid surface also indicated the lowest protein binding capacity for IgG (16.5 nmol/m2), Fc-IgG (32.4 nmol/m2) and HSA (16.7 nmol/m2), respectively. These imply that the PP-acrylic acid film is useful to fabricate as a low protein adsorption material which expected to decrease cell adhesion. Results of our investigation indicate that the plasma-polymerization technique is promising for fabrication of a smart NanoBio-interface which can control the protein adsorption on a solid-phase substrate using a suitable monomer such as allylamine for the large adsorption and acrylic acid for the small adsorption.

Introduction

Antibody immobilization is a necessary step to construct an immunoassay device. One immobilization method involves fixing via chemical reactions. However, this process is troublesome because of the need for indispensable chemical processes (Storri et al., 1998, Park et al., 2003). Another method involves a physical process, in which antibody is adsorbed spontaneously on the surface. Although this process offers ease of handling, we must select the material on which large amounts of antibody can be adsorbed to obtain high dose-response for immunoreaction because we can obtain antibody-bound material by simply immersing the plate into antibody solution (Muratsugu et al., 1993). Preparation of such a surface on which the activity of adhered antibodies can be retained will contribute to development of a new antibody technology; smart NanoBio-Interface techniques can achieve extra-high sensitivity and selectivity. On the other hand, the surface of suppression of non-specific adsorption of protein is expected to use as control of cell adhesion (Iwasaki et al., 1999). Therefore, in some case, the surface having high capacity of the protein binding is requested and in other cases, the opposite surface is needed.

Plasma polymerization techniques have proven to be useful for surface modification. They constitute an efficient tool for widely different monomers, even without regular functional groups associated with conventional polymerization on most substrates. Plasma polymerization has also been employed conventionally to fabricate thin functional films that can facilitate surface modification of materials. Plasma-polymerized films adhere firmly to most substrates and are highly resistant to chemical and physical treatments (Yasuda, 1985). Such films are expected to be useful as NanoBio-interface films that are firmly attached to transducers of chemical sensors, immunosensors, and immunoassay devices (Andrade, 1984; Sakurai et al., 1980, Mar et al., 1999, Kojima et al., 2003, Muratsugu et al., 1991, Muratsugu et al., 1997, Kurosawa et al., 2003, Kurosawa et al., 2004a).

This advantage of plasma polymerization has been applied to quartz crystal microbalance (QCM) by us (Kurosawa et al., 2003, Kurosawa et al., 2004a, Kurosawa et al., 2005). Because of its simplicity, convenience, low cost and real-time response, QCM devices have been examined in myriad fields for food analysis, clinical analysis, and environmental monitoring (Abad et al., 1998, Aizawa et al., 2001, Chou et al., 2002, Park et al., 2006, Kurosawa et al., 2005). In addition, QCM device has been used for immunosensor (Morgan et al., 1996, Bunde et al., 1998). The QCM detection principle is reliant upon recording the frequency decrease corresponding to a mass increase on the chip surface during biological interaction (Sakai et al., 1995, Bunde et al., 1998, Kurosawa et al., 2004b). Antibody must be immobilized on the crystal surface to capture the target compound, i.e. antigen, to implement a QCM-immunosensor. Normally, it was considered with no doubt that affinity of antibody compared with natural states was reduced considerably for a corresponding antigen when it was used for in vitro test. After all, this result can give reduced sensitivity of all in vitro immunoassays, especially immunosensors. Affinity of the immobilizing antibody should be assessed in order to construct a highly sensitive immunosensor with an antibody-modified surface.

Therefore, our previous paper intended to find the method in which the silver or gold electrode surfaces of QCM device were modified by the plasma-polymerized film so that a large amount of antibody adsorbed spontaneously. For this end, in our previous paper, the plasma-polymerized films were formed on silver plate (Kurosawa et al., 1991). However, when we want to show the general usage and applicability of the plasma-polymerized film to various devices such as NanoBio device, we should employ glass plates as substrates, on which various plasma-polymerized films were synthesized. Subsequently, their relative capacities as protein adsorbents were examined. The characterization of films formed was done and the surface properties such as the contact angle were determined. This study specifically addresses the surface modification process required for control of the adsorption amount of antibody on the substrate by the plasma-polymerization technique. We found that, among the films tested, PP-allylamine film showed remarkably high binding capacity for F(ab′)2-IgG, and PP-acrylic acid film showed lowest binding capacity for all tested proteins.

Section snippets

Materials

The following materials were obtained as indicated: Na125I (Amersham Biosciences, UK); HSA and human IgG (Sigma Chemical Co., USA); goat IgG Fc fragment (Fc-IgG) (Jackson Immunoresearch Laboratories Inc., USA); goat IgG F(ab′)2 fragment (F(ab′)2-IgG) (Cappel, USA); glass plates (5 mm × 7 mm × 0.15 mm; Matsunami Glass Ind. Ltd., Japan); polystyrene non-treated plates (5 mm × 5 mm × 1.35 mm; Eiken Chemical Co. Ltd., Japan); polyvinylchloride plates (5 mm × 5 mm × 1 mm) (Kasai Sangyou, Japan); and

Plasma polymerization

Monomers used for plasma polymerization were acrolein, acrylic acid, allylcyanide, and allylamine. These selected monomers are typical monomers having a functional group for plasma polymerization. Previously, we succeeded in forming PP-films from these monomers on Ag metal surface, (Kurosawa et al., 1991). As is the same as those in previous paper, the obtained films in this paper were insoluble in water, methyl alcohol, ethyl alcohol or acetone, which are good solvents for these monomers. The

Conclusion

Plasma-polymerized films were formed on flat glass plates using allylamine, acrylic acid, acrolein, and allylcyanide as monomers. Adsorption of immunoglobulin G (IgG), F(ab′)2-IgG, Fc-IgG and HSA was measured on these PP-films and on commercially available polymer plates. The adsorption isotherm followed the Langmuir equation, from which the binding constant and amount of saturation binding were estimated. We found that, in general, a cationic surface has higher affinity for protein adsorption

Acknowledgements

This work was supported financially in part by a Grant-in-Aid for Scientific Research on Priority Areas (16040217) from MEXT, for “Application of permselective and biocompatible membranes for improvement of quartz crystal microbalance biosensors” from the Daiwa Anglo-Japanese Foundation, and for “Study on monitoring of environmental risk compounds such as dioxins and endocrine disruptors using sensing systems” from the Ministry of the Environment, Japan.

References (28)

  • H. Aizawa et al.

    Sens. Actuators B

    (2001)
  • K. Al-Malah et al.

    J. Colloid Interface Sci.

    (1995)
  • R.L. Bunde et al.

    Talanta

    (1998)
  • S. Kurosawa et al.

    Biosens. Bioelectron.

    (2004)
  • S. Kurosawa et al.

    Biosens. Bioelectron.

    (2004)
  • M.N. Mar et al.

    Sens. Actuators B

    (1999)
  • M. Muratsugu et al.

    J. Colloid Interface Sci.

    (1991)
  • J.W. Park et al.

    Sens. Actuators B

    (2003)
  • G. Sakai et al.

    Sens. Actuators B

    (1995)
  • S. Storri et al.

    Biosens. Bioelectron.

    (1998)
  • J.M. Abad et al.

    Anal. Chem.

    (1998)
  • J.D. Andrade

    Surface and Interfacial Aspects of Biomedical Polymers

    (1984)
  • S.F. Chou et al.

    Clin. Chem.

    (2002)
  • J. Hiraoka et al.

    Kobunshi Ronbunshu

    (1985)
  • Cited by (0)

    This article is part of the Microsensors 2006 Special Issue which has now been published in Biosensors and Bioelectronics [Volume 22, Issue 4].

    View full text