A Novel Method of Immobilizing Antibodies on a Quartz Crystal

May 15, 1996 - Orientation and characterization of immobilized antibodies for improved immunoassays (Review). Nicholas G. Welch , Judith A. Scoble , B...
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Anal. Chem. 1996, 68, 1695-1700

A Novel Method of Immobilizing Antibodies on a Quartz Crystal Microbalance Using Plasma-Polymerized Films for Immunosensors Keijiro Nakanishi, Hitoshi Muguruma, and Isao Karube*

Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan

A novel method of immobilizing antibodies on quartz crystals for use in immunosensors was developed using an ethylenediamine plasma-polymerized film matrix. The films formed on the quartz crystals are extremely thin and homogeneous, and they incorporate amino groups. Sensors produced using this method are more reproducible from sample to sample and exhibit lower noise and higher sensitivity than sensors made using conventional immobilization methods, e.g., via polyethylenimine and (γ-aminopropyl)trimethoxysilane. This is, to our knowledge, the first reported application of plasma-polymerized films to quartz crystal microbalance immunosensors. Results on orientation-controlled immobilization of antibodies, reusability and calibration tests are also presented. Established immunoassay techniques, such as radio immunoassay, fluoroimmunoassay, enzyme immunoassay, and latex immunoassay, are widely used but suffer from disadvantages including complex, time-consuming procedures and potentially hazardous or expensive materials. In the last decade, many reports have been published on the use of quartz crystal microbalance (QCM) devices as sensors for a wide range of applications in the food industry,1,2 environmental monitoring, biotechnology, and clinical diagnostics.3,4 QCM biosensors offer advantages such as real-time output, high sensitivity and specificity, simplicity of use, and cost-effectiveness. QCM sensors are based on measurement of small mass changes on the surface of a quartz crystal caused by the specific adsorption of molecules by a specially modified surface. The potential of QCM devices in chemical sensor applications was realized after Sauerbrey derived the frequency-to-mass relationship,8

∆F ) (-2.3 × 106)F2∆M/A

(1)

where ∆F (Hz) is the change in the fundamental frequency of the coat crystal, F (Hz) is its resonant frequency, A (cm2) is the area coated, and ∆M (g) is the mass deposited. (1) Prusak-Sochaczewski, E.; Luong, J. H. T.; Guilbault, G. G. Enzyme Microb. Technol. 1990, 12, 173. (2) Plomer, M.; Guilbault, G. G.; Hock, B. Enzyme Microb. Technol. 1992, 14. (3) Muramatsu, H.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1986, 188, 257. (4) Muramatsu, H.; Tamiya, E.; Karube, I. Anal. Lett. 1989, 22, 2155. (5) Konig, B.; Gratzel, M. Anal. Chim. Acta 1993, 276, 329. (6) Konig, B.; Gratzel, M. Anal. Chim. Acta 1993, 281, 13. (7) Konig, B.; Gratzel, M. Anal. Lett. 1993, 26, 1567. (8) Sauerbrey, G. Z. Z. Physik. 1959, 155, 206. S0003-2700(95)00756-6 CCC: $12.00

© 1995 American Chemical Society

Techniques for depositing thin organic films are an important part of fabricating antibody-immobilized sensors. Antibody immobilization is usually achieved using polyethylenimine1,2,5-7 or (γ-aminopropyl)triethoxysilane1-7 matrices. However, those methods require a drying stage in air and a washing stage to remove excess unbound material after the quartz crystal is immersed in a reagent solution. If the duration and intensity of the washing steps are not kept uniform, the quantity of immobilized antibodies on each quartz crystal may vary due to variability in the mass of organic material immobilized on the quartz crystals. Excessive organic film material may also contribute to instability of frequency response. It is difficult to control the quantity of immobilized antibody on the quartz crystals, so it is desirable that a simple and uniform immobilization method be developed to facilitate mass production of QCM immunosensors. Immobilization via protein A is a good immobilization technique in terms of homogeneity and reproducibility from sample to sample but cannot be used to make reusable sensors because the protein A is removed from the quartz crystal when the binding antibodies are washed. Suri et al.9 reported that, although the protein A adsorbed on a gold surface of a quartz crystal was barely removed by only one treatment of 0.2 M glycineHCl buffer at pH 2.4, repeat treatments gradually removed the protein A from the gold surface. For example, the response had a decrease of ∼65% with four circle treatments of glycine-HCl buffer.9 Plasma-polymerized films offer a new alternative.10,11 Polymer film formation is achieved using glow discharge or a plasma of organic vapors. This process has some advantages, including the following: (1) an extremely thin, adherent organic film (