Anal. Chem. 2000, 72, 3805-3811
Oriented Immobilization of Antibodies onto the Gold Surfaces via Their Native Thiol Groups Arkady A. Karyakin,* Galina V. Presnova, Maya Yu. Rubtsova, and Alexey M. Egorov
Faculty of Chemistry of M.V. Lomonosov Moscow State University, 119899 Moscow, Russia
The general approach for site-oriented immobilization of antibodies onto gold supports is reported. The immobilization is carried out using the native sulfide groups of immunoglobulin (IgG). To liberate the thiol groups, the intact IgG was split into two half-IgG fragments without destruction of the binding site of the antibody. The immobilization of half-IgG fragments on the gold surface was carried out by simple adsorption. The antigen binding capacity of the half-IgG modified gold supports is similar to that of the gold surfaces with the traditionally linked antibodies and is much higher than for nonspecifically adsorbed intact IgGs. The immobilized antibodies, according to the proposed approach, maintain high antigen binding constants. The immobilization procedure provides orientation of IgG fragments in terms of the similar distance between the binding site of the antibody and the surface of the gold support, which does not cause the distribution of the apparent affinity constants. The high operational stability of half-IgG modified gold electrodes makes them applicable for analytical applications. The development of immunoassays during the past decades has revolutionized the determination of drugs and hormones in clinical and pharmaceutical chemistry as well as contaminants in the environmental area. Immunosensors, in which antibodies or antibody fragments are immobilized onto electrodes, on optical fibers or semiconductor chips, represent their promising application. Immunosensors can be divided into several classes depending on the transducer technology employed: piezoelectric,1, optical,2 and electrochemical.3 Obviously the development of an immunosensor requires immobilization of antibody (or in some cases antigen) on the transducer surface. Numerous coupling strategies have been applied for immobilizing antibodies onto different solid surfaces using defined linkages such as glutaraldehyde, carbodiimide, and other reagents such as succinimide ester, malein, and periodate.4,5 The direct attachment of antibodies to the gold surface seems to be the most important for immunosensors. Indeed, the surface of piezo and optic (surface plasmon resonance) transducers are * Corresponding author. Fax: (7-095) 939 2742. E-mail:
[email protected]. (1) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (2) Chaiken, I.; Rose, S.; Karlsson, R. Anal. Biochem. 1992, 201, 197-210. (3) Berggren, C.; Johansson, G. Anal. Chem. 1992, 69, 3651-3657. (4) Marco, M.-P.; Gee, S.; Hammock, B. D. Trends Anal. Chem. 1995, 14, 415425. (5) Marco, M.-P.; Gee, S.; Hammock, B. D. Trends Anal. Chem. 1995, 14, 341350. 10.1021/ac9907890 CCC: $19.00 Published on Web 07/21/2000
© 2000 American Chemical Society
covered by gold layers. Gold electrodes are also very useful in electrochemical immunosensors because they are chemically inert and have a prolonged double-layer potential region in aqueous solutions.6 In general, the known strategies for immobilization of biomolecules onto the gold surfaces are based on the strong attachment of thiol functional groups (SH or SS) to Au. The antibodies are usually linked to gold substrates using a bifunctional reagent with the thiol group on one side.7-10 However, the known methods of immobilization of antibodies onto the solid supports resulted in random orientation of biomolecules on the surface. The immobilization affects the specific binding constants because the apparent constants are dependent on the orientation of biomolecule on the solid support. Obviously, in case of random orientation, the distribution of the specific binding constants is achieved. Moreover, the loss of biological activity of antibodies upon immobilization, noticed in many cases,11 is associated with the random orientation of asymmetric macromolecules on the support surface.12 Thus, the oriented immobilization of antibodies onto the gold supports is an extremely important problem of immunochemistry and immunosensors. To develop a general approach for oriented immobilization of antibodies, let us consider the structure of immunoglobulins.13 Immunoglobulin G (IgG) is a special class of glycoproteins containing two couples of polypeptide chains. Each couple contains the heavy chain (H) and the light chain (L) (Figure 1). The heavy chain contains one variable region and three constant regions: CH1, CH2, and CH3. The light chain consists of one constant and one variable region denoted by CL and VL, respectively. The two variable regions of heavy and light chains are involved in the binding site of the antibody. The two heavy chains are linked via disulfide bonds. The amount of disulfide bonds between the heavy chains is dependent on the type of immunoglobulin. It is possible to split two heavy chains by reducing immunoglobulin with 2-mercaptoethylamine and to obtain the two “half(6) Hamelin, A. Double-layer properties at sp and sd metal single-crystal electrodes. In: Modern Aspects of Electrochemistry, Conway, B. E.; White, R. E.; Bokris J. O′M. Eds.; Plenum Press: New York & London; 1985, 16, 1-. (7) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosensors & Bioelectronics 1997, 12, 977-989. (8) You, H. X.; Lin, S.; Lowe, C. R. Micron 1995, 26, 311-315. (9) You, H. X.; Disley, D. M.; Cullen, D. C.; Lowe, C. R. Micron 1995, 26, 121132. (10) Lotzbeyer, T.; Schuhmann, W.; Schmidt H.-L. J. Electroanal. Chem. 1995, 395, 341-344. (11) Spitznagel, T. M.; Clark, D. S. Biotechnology 1993, 11, 825-829. (12) Lu, B.; Smyth, M. R.; O′Kennedy, R.; Analyst 1996, 121, 29R-32R. (13) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Microchimica Acta 1998, 128, 127-143.
Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3805
Figure 1. Scheme of the site-oriented immobilization of antibodies onto the gold surface using their native thiol groups.
immunoglobulin” fragments (Figure 1).14 The procedure is expected to not affect the binding site of the antibody, which is formed by the coupling of heavy and light chains. We report the general approach for site-oriented immobilization of immunoglobulins onto the gold supports (Figure 1, Table 1). The immobilization is carried out using the native immunoglobulin thiol groups which are liberated after the splitting of the intact antibody into two half-IgG fragments without the destruction of the binding site of the antibody. The half-IgG fragments were immobilized onto gold supports by simple adsorption. The proposed approach is advantageous over existing methods because the immobilized antibodies maintain both high antigen binding constants and high stability. The immobilization procedure provides orientation of IgG fragments so that there is a similar distance between the binding site of the antibody and the electrode surface, which does not cause the distribution of the apparent affinity constants. EXPERIMENTAL SECTION Reagents. All solutions were prepared with Millipore (MilliQ) water. Inorganic chemicals, N-acetyl cysteine and ethylendiaminetetraacetic acid (EDTA), were purchased from Sigma (St. Louis, MO). Sephadex and BrCN-Sepharose were purchased from Pharmacia (Uppsala, Sweden). Horseradish peroxidase (HRP) (RZ 3.3) was purchased from Biozyme (Blaenavon, UK). Mouse monoclonal antibodies PO1 and rabbit polyclonal antibodies specific to horseradish peroxidase, as well as specific antibodies against mouse and rabbit immunoglobulins, were provided by Immunotech (Moscow, Russia). 5,5′-dithiobis (2-nitrobenzoic acid) was purchased from Aldrich (Steinheim, Germany).
Instrumentation. Electrochemical measurements were carried out using a 641 VA Detector (Metrohm, Switzerland). Solution flow was maintained by a Masterflex 7524-15 peristaltic pump (Cole Parmer, Vernon Hills, IL). Chromatographic columns were purchased from Pharmacia (Uppsala, Sweden), a UV recorder from LKB (Uppsala, Sweden), a spectrophotometer DU-8 from Beckman (Fullerton, CA), electrophoresis equipment from BioRad (Philadelphia, PA). Luminometer LumiFlow was provided by Immunotech (Moscow, Russia). Electrochemical Methods. The flow-through cell designed15 was of the confined-wall-jet type. The inlet section of the cell contained the Ag/AgCl reference electrode in a separate circular chamber filled with 0.1 M KCl from an external syringe. This chamber contacted the working electrode space by four holes (0.3 mm) concentrically surrounding the inlet (L, 0.5 mm). The auxiliary electrode was a platinum wire encircling the outlet chamber. The gold disk electrodes (L, 1.0 mm) were used as working electrodes. Prior to use, the gold electrodes were cleaned in a 1:1 mixture of concentrated sulfuric acid and hydrogen peroxide and polished with 1-µm alumina. The distance between the nozzle and working electrode was about 2 mm. Pump pulsation damping was provided by a 5-m length of Teflon tubing (L, 0.5 mm) placed before the cell. Purification of Antibodies. An IgG fraction was precipitated with sodium sulfate (0.18 g per 1 mL of antibody solution) and then dialized against PBS (0.01 M KH2PO4/KHPO4 + 0.15 M NaCl, pH 7.4) at 4 °C overnight. The affinity purification was carried out as follows. Peroxidase (10 mg) was dissolved in buffered water solution of 0.1 M NaHCO3 and 0.5 M NaCl. BrCN activated Sepharose 4B powder (1.1 g) was washed on a sinter-
(14) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized affinity ligands technique 1992, Academic Press: San Diego.
(15) Appelqvist, R.; Marko-Varga, G.; Gorton, L.; Torstensson, A.; Johansson G. Anal. Chim. Acta 1985, 169, 237-247.
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Table 1. Catalytic Activities of the Gold Supports Modified with the Enzyme Peroxidase Using Different Immobilization Schemesa
* The values are the mean of not less than 5 experiments; the Standard deviations (relative to the mean value) were
