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Atomic Force Microscopic Study of Specific Antigen/ Antibody Binding M. E. Browning-Kelley,† K. Wadu-Mesthrige,‡ V. Hari,*,† and G. Y. Liu*,‡ Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, Michigan 48202 Received September 20, 1996X Using atomic force microscopy (AFM), we systematically studied the binding of three pairs of specific antigen/antibody systems: bovine serum albumin, tobacco etch virus capsid protein, and tobacco mosaic virus capsid protein and their respective specific antibodies. Our goals were to find a substrate for antigen immobilization, characterize individual antigen/antibody complexes, and investigate the antigen/antibody binding process. We found that the antigen protein can be immobilized on a -COOH-terminated selfassembled monolayer surface. Individual antigens and antigen/antibody complexes are easily identified from AFM images taken in liquid or under ambient laboratory conditions. The in situ studies suggest that antibody-antigen reactions occur in less than 4 min in buffer and that the reaction complexes are stable adsorbates once formed. As control experiments, nonspecific antibodies of equal and higher concentrations than those of specific antibodies have been used. There was no binding between nonspecific antibodies and the antigen-immobilized surfaces. The experimental results suggest that the procedure established here may be used for specific antibody detection. In addition, this study has also enhanced our understanding of antigen/antibody binding processes.

Introduction A molecular level understanding of specific antigen/ antibody interactions is fundamentally important in the life sciences. Atomic force microscopy (AFM) provides a promising technique to address this issue, since this method can operate under physiological conditions and enables high-resolution microscopic images to be acquired.1-6 While several successful experiments have been carried out using AFM to visualize the topography of antibodies or antigen/antibody complexes7-11 and to measure the antigen/antibody binding force,12,13 there are few reports of in situ studies of the antigen/antibody binding process. Several technical challenges hinder in situ studies and the development of AFM into a more widely adopted tool in immunodiagnostics. First, antigen or antibodies must be immobilized on the surface in order to conduct AFM studies. Such immobilization needs to be strong enough for AFM imaging, and at the same time, without altering †

Department of Biological Sciences. Department of Chemistry. X Abstract published in Advance ACS Abstracts, January 1, 1997. ‡

(1) Binning, G.; Quate, C. F.; Berger, C. Phys. Rev. Lett. 1986, 56, 930. (2) Marti, O.; Amrein, M. Eds. STM and SFM in Biology; Academic Press: San Diego, 1993. (3) Yang, J.; Tamm, L. K.; Somlyo, A. P.; Shao. Z. J. Microsc. (Oxford) 1993, 171, 183. (4) Allen, M. J.; Bradbury, E. M.; Balhorn, R. J. Struc. Biol. 1995, 114 (3), 197. (5) Muller, D. J.; Schabert, F. A.; Buldt, G.; Engel, A. Biophys. J. 1995, 68, 1681. (6) Karraasch, S.; Hegerl, R.; Hoh, J. H.; Baumeister, W.; Engel, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 836. (7) Huber, W.; Richmond, T.; Anselmetti, T.; Schlatter, D.; Dreier, M.; Frommer, J.; Guntherodt, H.-J. Nanobiology 1994, 3, 189. (8) Mazeran, P. E.; Loubet, J. L.; Martelet, C.; Theretz, A. Ultramicroscopy 1995, 60, 33. (9) Ohnesorge, F.; Heckle, W. M.; Haberle, W.; Pum, D.; Sara, M.; Schindler, H. Schilcher, K.; Kiener, A.; Smith, D. P. E.; Sleyter, U. B.; Binning, G. Ultramicroscopy 1992, 42-44, 1242. (10) Quest, A. P.; Bergman, A. A.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Scanning Microsc. 1995, 9 (2), 395. (11) Muller, D. J.; Schoenenberger, C-A.; Buldt, G.; Engle, A.; Biophys. J. 1996, 70, 1796. (12) Hinterdorfer, P.; Baumagartner, W.; Gruber, H.-J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (13) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntheroid, H-J. Biophys. J. 1996, 70, 2437.

the bioreactivity of antigens or antibodies. Second, the substrate surface morphology and reaction conditions (e.g., buffer concentration) must be carefully controlled to avoid interference with the AFM image contrast. Third, the AFM scanning head must be stable enough so that images can be taken during and after the injection of reactants, e.g., antibody solution. Protein immobilization on surface is a very active area of research. The strategies reported in the literature for protein immobilization include modification of substrate surfaces, modification of the protein adsorbates, and a combination of both modifications. Examples of surface modifications include glow discharge of graphite, and chemical functionalization of mica, silica, or gold surfaces using self-assembly or Langmuir-Blodgett techniques.14-18 Protein modifications can be achieved chemically, for example, by treatment with phenylglyoxal followed by NaBH4.19 Other attempts to improve the protein adhesion to surface involve combinations of both surface and protein modification, for example, by the use of the biotin/ streptavidin interaction.20 Recently, a photoactive selfassembled monolayer (SAM) on gold was used to attach proteins.21 The philosophy behind our approach was to use the minimum amount of surface and protein modifications and, at the same time, achieve optimized immobilization without changing the bioreactivity of the adsorbate (14) Walivaara, B.; Warkentin, P.; Lundstrom, I.; Tengvall, P. J. Colloid Interface Sci. 1995, 174, 53. (15) Vinckier, A.; Heyvaert, I.; Dhoore, A.; Mckittrick, T.; Vanhaesendonck, C.; Engelborghs, Y.; Hellemans, L. Ultramicroscopy 1995, 57 (4), 337. (16) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052. (17) Weisenhorn, A. L.; Gaub, H. E.; Hansma, H. G.; Sinsheimer, R. L.; Kelderman, G. L.; Hansma, P. K. Scanning Microsc. 1990, 4, 511. (18) Wagner, P.; Kernen, P.; Hegner, M.; Ungewickell, E.; Semenza, G. FEBS Lett. 1994, 356, 267. (19) Droz, E.; Taborelli, M.; Descouts, P.; Wells, T. N. C. Biophys. J. 1994, 67, 131. (20) Davies, J.; Roberts, C. J.; Dawkes, A. C.; Sefton, J.; Edwards, J. C.; Galsbey, T. O.; Haymes, A. G.; Davies, M. C.; Jackson, D. E.; Lomas, M.; Shakesheff, K. M.; Tendleer, S. J. B.; Wilkins, M. J.; Williams, P. M. Langmuir 1994, 10, 2654. (21) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Micheal, B.; Gerber, Ch.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997.

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systems. Instead of using specific interactions, such as, streptavidin-biotin pairs or covalent binding,14-21 we chose to use -COOH-terminated SAM on ultraflat gold22,23 as the “universal protein immobilizer” for this study. As demonstrated below, such a surface satisfies the above requirements. Ex situ AFM studies were first conducted to visualize the static structure of the antigen proteins and the antigen/ antibody complexes. These ex situ studies also helped us to determine the optimum conditions for antigen/antibody reactions, such as concentration of antigens and antibody, incubation time, etc. Once the reaction conditions were established, the antigen/antibody binding processes were monitored in situ, in real time, and under a phosphatebuffered saline (PBS) solution. Experimental Section A. Preparation and Purification of Antigen and Antibody. Three antigen proteins were used for this study: bovine serum albumin (BSA), tobacco etch virus capsid protein (TEVCp), and tobacco mosaic virus capsid protein (TMVCp). The BSA, fraction V, which is essentially fatty acid free, was purchased from Sigma Chemicals. Both the TEV and TMV capsid proteins were first extracted from virus particles that had been isolated from plants according to established protocols.24,25 The viral capsid proteins (Cp) were then isolated from these viruses using the Fraenkel-Conrat method.26 The purified proteins were stored at 4 °C in 20 mM Tris buffer (pH 7.5) until needed. All three antigens were fractionated through a Sephadex G7550 (Pharmacia) column in order to remove large aggregates that formed during storage. Chromatographic fractions consisting mainly of monomeric protein were selected for AFM sample preparations. After fractionation, the concentration of the protein monomeric fraction was calculated by absorbance spectroscopy at OD 280 nm. The initial concentrations for BSA, TEVCp, and TMVCp were 100, 1.66, and 0.14 mg/mL, respectively. The three specific antibody (IgG) species were purified rabbit IgG fractions. The anti-BSA IgG was obtained from Sigma Chemicals (stock concentration of 17.96 mg/mL). The antiTEVCp IgG was obtained from Agdia (stock concentration of 2.0 mg/mL). The anti-TMVCp IgG protein (stock concentration of 1.6 mg/mL) was purified by protein A affinity chromatography.27 The nonspecific (normal) rabbit IgG used for control experiments was obtained from Sigma Chemicals (stock concentration of 10 mg/mL). B. Preparation of the Substrate. Two important criteria for the substrate preparation in this AFM study are (1) the protein affinity to the substrate must be adequately strong so that the antigens can be immobilized on surface without sacrificing their bioreactivity and (2) the substrate should be smooth enough so that proteins can easily be identified from AFM topographies. Mica surface is the most commonly used substrate for protein adsorption because it is hydrophilic and atomically flat.28,29 However, the binding of our antigen proteins to mica was relatively weak; as a result, the proteins were mobile on the surface under our AFM imaging conditions. To increase the affinity of proteins to surfaces, -COOH-terminated self-assembled monolayers were used. The substrate, ultraflat gold thin film, 1500 Å in thickness, was first prepared according to (22) Hegner, M.; Wagner, P.; Semenza, G. Surf. Sci. 1993, 291, 39. (23) Wagner, P.; Hegner, M.; Guntheroid, H-J.; Semenza, G. Langmuir 1995, 11, 3867. (24) Rochon, D.; In Vivo and In Vitro Encapsidation of Host RNA by TMV coat Proteins. Ph.D. Thesis, Wayne State University, Detroit, MI, 1985. (25) Hari, V.; Siegel, A.; Rozek, C.; Timberland, W. E. Virology 1979, 92, 568. (26) Fraenkel-Conrat, H.; Singer, B. Biochem. Biophys. Acta 1957, 24, 540. (27) Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moorc, D. D.; Seidman, J. G.; Smith, J. A.; Struhl, K. Eds. Current Protocols in Molecular Biology; Wiley-Interscience: New York, 1987; Vol. 2. (28) Wadu-Mesthrige, K.; Pati, B.; McClain, W. M.; Liu, G.-Y. Langmuir 1996, 12, 3511. (29) Yang, J.; Tamm, L. K.; Tillack, T. W.; Shao, Z. J. Mol. Biol. 1993, 229, 286.

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Figure 1. Schematic diagram of protein immobilization and antigen/antibody complex formation. Although there are many possible binding orientations, only the most likely one is shown here. The dimensions of the antigens and primary and secondary antibodies are indicated in the drawing (not to scale), which will be used later as a reference to identify proteins from AFM images. a new procedure by P. Wagner et al.22,23 The resulting surface has a mean roughness as small as 2-5 Å, which is sufficiently smooth for AFM studies of most biosystems. After it was thoroughly rinsed with hexane, ethanol, and water, the gold thin film was immersed into a 1.0 mM HS(CH2)3COOH (Pfaltz and Bauer, Inc., 90%) solution. Thiols are known to form selfassembled monolayers with the -COOH terminal groups exposed at the interface.30-35 The choice of a -COOH-terminated surface was inspired by reports that certain carboxylated microspheres would adsorb proteins in the absence of coupling agents.36,37 It is commonly believed that the electrostatic interactions between -COOH groups and proteins are mainly responsible for the protein immobilization.36 C. Sample Preparation for AFM Studies. The first step was to immobilize the antigens on the -COOH-terminated substrate. Twenty microliters of the respective antigens at concentrations of 7.9, 50.0, and 36.0 µg/mL for TEVCp, BSA, and TMVCp, respectively, were deposited on the modified substrate and allowed to dry in air. These concentrations were chosen because they corresponded to approximately a monolayer coverage. Immediately after solvent evaporation, the antigen layer was washed three times with water to remove any residual salt deposits or loosely adsorbed proteins. The samples were then allowed to dry completely before imaging. Ideally, antibodies should recognize the adsorbed antigens as illustrated in Figure 1. In reality, the amount of antibody binding depends on several factors, including the orientation of adsorbed antigen, concentration of antibody, and other incubation conditions. The conditions of primary IgG reactions with surfaceadsorbed antigens are summarized in Table 1. For the ex situ studies, the IgG in solution was applied to the antigen-covered surface and allowed to incubate in a humid chamber at room temperature for a specified period of time. The IgG solution was drawn off with a pipette, and the surface was washed three times with water to remove any weakly adsorbed IgG. For the TMVCp system, we also reacted the primary IgG with a second IgG. The secondary IgG (goat antirabbit (GAR) Sigma Biochemical) is specific for all rabbit IgG molecules and should (30) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 3665. (31) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 5897. (32) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 110, 6560. (33) Laibinis, P. E.; Whiteside, G. M. J. Am. Chem. Soc. 1992, 114, 1990. (34) Ulman, A. An Introduction to Ultrathin Organic Films From Langmuir-Blodgett to Self-Assembly; Academic Press, Inc.: San Diego, CA, 1991. (35) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (36) Yoon, J -Y.; Park, H -Y.; Kim, J -H.; Kim, W -S. J. Colloid Interface Sci. 1996, 177, 613. (37) Duke Scientific Corporation, Bulletin 88c; 1990.

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Table 1. Summary of the Reaction Conditions and the Resulting Coverage of Specific Antigen/Antibody Complexes antigen

IgG concn, µg/mL

incubtn time, h

coverage of IgGa %

BSA

anti-BSA

1.8 1.8 5.4 5.4

0.5 1.5 0.5 1.5

62 ( 1.7 65 ( 2.2 85 ( 3.2 85 ( 2.1

TEVCp

anti-TEV

0.4 0.4 1.2 1.2

0.5 1.5 0.5 1.5

12 ( 4.0 12 ( 3.8 43 ( 2.0 45 ( 1.0

TMVCp

anti-TMV

7.2

0.5

55 ( 1.0

a Value reported here is the mean coverage extracted from AFM images taken from different areas of the sample.

Figure 2. Schematic diagram of the experimental setup used for in situ AFM imaging under liquid conditions. Using this setup, liquid can be injected into or removed from the liquid cell without obvious perturbation to the system. therefore bind to any primary IgG that it contacts. Following the procedure of primary antibody incubation, 50 µL of GAR IgG (1:10000 dilution) in PBS (pH 7.3, 140 mM NaCl, 3.0 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) was applied to the antiTMVCp-covered surface. In the control experiments, nonspecific IgG was used with the same incubation times and at the same or much higher concentrations (20 µg/mL) than those of the specific antibodies. D. Atomic Force Microscopy. The AFM used for this study was an in-house-constructed state-of-the-art microscope, which allows contact and non contact mode image, multichannel data acquisition, and operates under ambient laboratory conditions, in vacuum, or in solution.38,39 The cantilevers were sharpened microlevers from Park Scientific, Inc., with a force constant of 0.1 N/m, or from Digital Instrument with a force constant of 0.58 N/m. A quadrant photodiode detector allows simultaneous measurement of both topography and friction. Two series of AFM experiments were carried out. The first addressed the static structures of the bioadsorbates after each step of sample preparation (see Figure 1). The second were in situ studies of the three antigen/antibody reactions. The structure characterization was accomplished using AFM in the contact mode under ambient laboratory conditions of constant force (load). The magnitude of the imaging force is determined from the force curve and includes both the capillary contribution and the force acting on the surface due to cantilever bending. A systematic study was conducted to investigate the antigen/ antibody reactions under different conditions, as summarized in Table 1. Once the optimum conditions were established, the antigen/antibody reactions were then monitored in situ using the setup illustrated in Figure 2. In this setup, AFM images can be taken immediately after the injection of IgG solution. (38) Kolbe, W. F.; Ogletree, D. F.; Salmeron, M. Ultramicroscopy 1992, 42-44, 1113. (39) Liu, G. Y.; Fenter, P.; Chidsey, C. E. D.; Ogletree, D. F.; Eisenberger, P.; Salmeron, M. J. Chem. Phys. 1994, 101, 4301.

Imaging in liquids, such as water, prevents the capillary interaction between the AFM tip and the sample surface.40,41 In addition, much gentler forces (0.1 nN) were used during imaging, to reduce the perturbation to the adsorbates. Therefore, higher resolution AFM images can be obtained under liquid conditions.40,41 Furthermore, one can adjust the solution and mimic physiological conditions. For the in situ studies, the antigens were first imaged in water. The IgG in PBS (pH 7.3, 140 mM NaCl, 3.0 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) was then injected into the AFM liquid cell. The change in surface morphology was then monitored in situ and in real time. From the changes of the AFM images, one can extract information on antigen/antibody binding processes.

Results and Discussion A. BSA/Anti-BSA IgG Systems. We chose BSA to begin our investigation because of its simple structure and near spherical shape. Figure 3 shows the topographic images of BSA on HOOC(CH2)3S/Au and the surface topographies after incubation with nonspecific IgG and specific anti-BSA IgG solutions, respectively. As shown in Figure 3a, BSA forms a monolayer under our deposition conditions. By using a higher imaging force of 12.5 nN, we were able to remove some BSA particles from their adsorption sites and image the underlying substrate. This “displacement” experiment allows the measurement of the height of each BSA particle more precisely. The height of individual BSA particles measured from Figure 3a and measured from the displacement experiment is 34 ( 2 Å, which is consistent with the theoretical value of 40 Å.42 The width of the individual BSA particles measured from Figure 3a is 68 ( 5 Å. The slightly compressed height and broadened width of the BSA are due to deformation of these molecules by the tip during imaging and convolution of the tip geometry.43-45 Figure 3b exhibits the same surface morphology as Figure 3a, which indicates that there is no apparent binding between nonspecific IgG and BSA. In contrast, many new and brighter “spots” appeared after the BSA was incubated with anti-BSA IgG (Figure 3c). The height of these bright spots is 100 ( 5 Å which corresponds very well to the dimensions of individual BSA/ anti-BSA complexes. Immunoglobulins (IgG) are large proteins composed of two heavy chains (MW 50 kD) and two light chains (MW 25 kD) attached by disulfide bridges that form a Y-like structure. The width and height are 145 and 85 Å, respectively, as determined by X-ray diffraction.46 To further verify the height of the proteins, we again removed the adsorbates under a high imaging force of 26 nN. The depth of the protein layers measured for BSA and BSA incubated with nonspecific IgG is 34 ( 2 Å, which corresponds to those of the adsorbed BSA particles. In contrast, the total height is 100 ( 10 Å in Figure 3c, which is consistent with the BSA/anti-BSA IgG complexes. The above observations suggest that the -COOHterminated surface allows for the adequate immobilization of proteins. The individual BSA proteins, as well as the BSA/anti-BSA IgG complexes, can be easily identified from the topographic images. (40) Weisenhorn, A. L.; Hansma, P. K.; Albrecht, T. R.; Quate, C. F. Appl. Phys. Lett. 1989, 54, 2651. (41) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannel, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989, 243, 158. (42) Rosenoer, V. M.; Oratz, M.; Rothschild, M. A. Albumin Function and Uses; Pergamon Press: New York, 1977. (43) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 115. (44) Zenhausern, F.; Adrian, M.; Emch, R.; Taborelli, M.; Jobin, M.; Descouts, P. Ultramicroscopy 1992, 42-44, 1168. (45) Vensenka, J.; Guthold, M.; Tang, C. L.; Keller, D.; Delaine, E.; Bustamante, C. Ultramicroscopy 1992, 42-44, 1243. (46) Silvertone, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140.

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Figure 3. Topographic images (0.5 × 0.5 µm2 area) of immobilized BSA and BSA/anti-BSA IgG complexes taken in ambient laboratory conditions. (a) Three-dimensional displays of BSA adsorbed on the surface. The surface morphology changes very little after incubation in nonspecific antibody (b). However, antibody-antigen complexes are clearly visible in (c) after incubation in specific anti-BSA antibody solution.

Figure 4. In situ AFM images (both two dimensional and three dimensional) of BSA-immobilized surface before (top) and after anti-BSA IgG (bottom) injection. Four minutes after injection of anti-BSA IgG, antigen/antibody complexes appear as bright spots in the bottom image. The dimensions of BSA (top) and BSA/anti-BSA IgG (bottom), as shown in the cursor profiles, correspond well with the theoretical values.

In situ experiments were performed to gain a deeper understanding of the antigen/antibody reaction process. The BSA layer was first imaged under water (Figure 4a). Its surface morphology was very similar to that observed under ambient laboratory conditions (Figure 3a). The measured height and width of individual BSA particles are 38 ( 1 and 56 ( 4 Å, respectively. After an image area was selected, 5.4 µg/mL of anti-BSA IgG in 200 µL of PBS was injected into the liquid cell (see Figure 2). It should be noted that both IgG and PBS were diluted approximately 10-fold after injection, since the liquid cell contained water. Within 4 min, the surface morphology changed. As shown in Figure 4b, many bright spots appeared. The dimensions of these spots varied slightly

from one to another; however, the range was consistent with that of the expected individual BSA/anti-BSA IgG complexes. Surface coverage of the antibody-antigen complexes was about 25%, and these complexes were distributed randomly on the substrate. The surface morphology, as shown in Figure 4b, did not change with incubation time. This is consistent with the ex situ experiments summarized in Table 1, which shows that the surface coverage depends sensitively on antibody concentration but does not vary with the incubation time. (Maximum antibody coverage (reaction equilibrium) is reached within a few minutes.) Another interesting observation is the lack of BSA/anti-BSA IgG binding in the absence of a PBS buffer. To our knowledge, the

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Figure 5. AFM images of adsorbed TEVCp and TEVCp/anti-TEVCp IgG complexes taken in air. The images are displayed in two and three dimensions, respectively, along with the cursor profiles of typical adsorbates. (a) Surface is homogeneously covered by ellipsoidal particles with a diameter of 12 ( 6 Å. Each particle represents a TEVCp. As shown in b, incubation in nonspecific IgG does not results in any antigen/antibody bindings. Incubation in 0.4 µg/mL anti-TEVCp IgG for 30 min leads to the formation of antigen/antibody complexes as shown in c.

experiment described above and the subsequent experiments described here represent the first in situ AFM study of BSA and anti-BSA IgG binding. The kinetics observed here are in good agreement with previously described antibody-antigen reactions.47-49 B. TEVCp/Anti-TEVCp IgG Systems. A more complicated system, TEVCp/anti-TEVCp IgG, was also studied. Unlike the spherical shaped BSA, the shape of TEVCp resembles a slice of “cake”. The four sides of the quadrilateral are 8, 20, 20, and 30 Å, respectively, and the height is 24 Å.50 When adsorbed onto the HOOC(CH2)3S/ Au, these proteins may assume different orientations. AFM images in Figure 5a do suggest, although not very precisely, different geometries of the adsorbed TEVCp particles. (47) Borrebaeck, C. A. K.; Malmborg, A. C.; Furebring, C.; Michaelson, A.; Ward, S.; Danielson, S.; Ohlin, M. Biotechnology 1992, 10, 697. (48) Tanimoto, S.; Kitano, H. Colloids Surf., B 1995, 4, 259. (49) Nygren, H. Colloids Surf., B 1995, 4, 243. (50) The dimensions of TEVCp and TMVCp were estimated on the basis of the known dimensions and structures of the viruses. Both capsid proteins have a shape similar to that of a slice of cake.

As shown in Figure 5b, after incubation with nonspecific IgG, the TEVCp surface shows the same surface morphology as seen in Figure 5a, which indicates that there is no apparent binding between nonspecific IgG and TEVCp. In contrast, after incubation with specific antiTEVCp IgG, new and brighter “spots” appear, as shown in Figure 5c. The average height of these bright spots is 110 ( 20 Å, which indicates the formation of a TEVCp/ anti-TEVCp IgG complex. Longer incubation times have very little effect in terms of surface morphology. However, higher anti-TEVCp IgG concentrations leads to higher IgG coverage. In addition to individual TEVCp/antiTEVCp IgG complexes, some larger spots (dimensions: 400-600 Å in width and 100-80 Å in height) were also visible when the IgG concentration was 1.2 µg/mL (images not shown). These large spots correspond to side-by-side aggregation of IgG on the antigen-immobilized surface with approximately two-five IgG particles per aggregate. Our observation is consistent with previous reports that, at high concentrations, antibodies tend to aggregate on immobilized antigen surfaces.49,51

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Figure 6. In situ imaging of TEVCp/anti-TEVCp IgG complex formed in PBS solution. (a) Topographic image of TEVCp layer before the injection of anti-TEVCp IgG. (b) Topographic images taken 4 min after injection of anti-TEVCp IgG.

The variation in height of the individual TEVCp/antiTEVCp IgG complexes, as shown in the cursor plot of Figure 5c, is likely caused by the different orientations of the adsorbed TEVCp and antigen/antibody complexes. Figure 6 shows two selected images of the in situ study of the TEVCp/anti-TEVCp IgG reaction. Within 4 min after injection of the anti-TEVCp IgG (1.2 µg/mL in PBS) into the liquid cell, the surface morphology changed as shown. Individual TEVCp/anti-TEVCp IgG complexes appeared as bright spots in Figure 6b. Similar to the BSA/anti-BSA IgG binding process, 30% of the surface is covered by the TEVCp/anti-TEVCp IgG complexes, and the coverage does not vary with a subsequent increase in incubation time. C. TMVCp/Anti-TMVCp IgG System. TMVCp has a shape similar to that of TEVCp but with a larger total volume. Figure 7a-d shows the representative images of each step of the TMVCp/anti-TMVCp IgG reactions. Figure 7a is the topographic image of TMVCp immobilized on the substrate. A monolayer coverage of ellipsoidal objects can be seen. The height of individual particles measured from this image is 10 ( 8 Å, and their width is 60 ( 10 Å. After incubation of TMVCp with anti-TMVCp IgG, new bright spots appeared on the surface (Figure 7c). The measured sizes of these spots are 100 ( 25 Å in height and 175 ( 25 Å in width. These are consistent with the size for a TMVCp/anti-TMVCp complex. In this particular experiment almost all of the TMVCp/anti(51) Stenberg, M.; Nygren, H. J. Immunol. Methods 1988, 113, 3.

TMVCp IgG complexes appeared as individuals; no aggregation of IgG on the antigen-immobilized substrate was seen. Most immunoassays employ a secondary antibody that is conjugated with a detectable activity (alkaline phosphatase, horse radish peroxidase, etc.) in order to detect the presence of a primary antibody/antigen complex.27 As demonstrated in Figures 3-8, specific antigen/antibody bindings can be detected directly by AFM without introducing secondary antibodies. To satisfy our curiosity as to how the AFM images would change with the adsorption of a secondary antibody, we incubated the above described system (TMVCp/anti-TMVCp IgG complexes) with a solution of GAR IgG (previously described). New and much larger spots were clearly visible in Figure 7d. The heights of these particles were around 200-300 Å, which is comparable with the theoretical sum of the heights of the antigens, primary antibody, and secondary antibody. With the presence of the secondary antibody, the AFM image contrast is further enhanced. The in situ AFM experiments for TMVCp followed the same procedure as the TEVCp system. Figure 8a is the topographic image of TMVCp taken under water. The TMVCp surface morphology is similar to that observed under ambient laboratory conditions. After an image area was selected, 7.2 µg/mL of anti-TMVCp IgG in 50 µL of PBS was injected into the liquid cell (see Figure 2). Within 3 min, the surface morphology changed, as shown in Figure 8b. Anti-TMVCp/TMVCp IgG complexes appeared as bright spots in Figure 8b. Some aggregation of TMVCp

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Figure 7. AFM images of immobilized TMVCp and TMVCp/anti-TMV IgG complexes taken in ambient laboratory conditions. Images a and b are TMVCp adsorbed on the surface and after incubation with nonspecific IgG, respectively. Incubation in 7.2 µg/mL of anti-TMVCp results in the formation of antigen/antibody complexes. These complexes are randomly distributed on the surface as shown in c. Image d shows the adsorption of secondary antibody GAR IgG on the primary antibody (anti-TMVCp IgG) covered surface.

IgG on the antigen layer is also present in some areas as shown in Figure 8c. In summary, the work described above has demonstrated that a -COOH-terminated surface can effectively immobilize proteins without sacrificing the bioreactivity. The adsorbed proteins BSA, TEVCp, and TMVCp are sufficiently stable for AFM imaging even under a contact mode. The control experiments using nonspecific IgGs

have confirmed that binding only occurs between adsorbed antigens and the corresponding specific IgGs. The antigen/antibody binding process has also been monitored in situ via AFM in real time. Stable antigen/antibody complexes were observed in less than 4 min after the injection of an antibody, and such reactions cannot occur in the absence of buffer. The coverage of antigen/antibody does not vary with incubation time. However, both the

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Figure 8. In situ imaging of TMVCp/anti-TMV IgG reaction in PBS solution. Individual TMVCp particles are clearly visible in a taken before anti-TMV IgG injection. Antibody-antigen complexes are formed in less than 3 min after injection of anti-TMV IgG. As shown in b, most complexes are dispersed on the surface; however, aggregation does occur in some areas, as shown in image c.

coverage and aggregation status depend upon the antibody concentration. Four important features of this work need to be reiterated here: the simplicity of the substrate preparation, specific adhesion of the antibodies to the antigenimmobilized surface, the fast binding kinetics of antibodies to the antigen, and the ability to visualize individual antigens, antibodies, and antigen/antibody complexes. These results suggest that the AFM protocol described here may be developed further into an immunoassay for the detection of very low concentration’s of antibodies or antigens. The ability to do in situ imaging has deepened and will continue to enhance our understanding of the antigen/antibody reaction kinetics and mechanism. Work is in progress to study the antigen- antibody reaction of

animal antibodies, as well as antibody/cell and antibody/ virus interactions. Acknowledgment. We thank Ms. L. Deng for her help in preparation of TEVCp and Mr. Song Xu for his help in the preparation of self-assembled monolayers. G.Y.L. gratefully acknowledges the Camille and Henry Dreyfus Foundation for a New Faculty Award and the Beckman Foundation for a Young Investigator Award. This work is also supported by the Research Stimulation Fund and G.Y.L.’s startup fund of Wayne State University, the Institute for Manufacturing Research, and NSF Grant CHE- 9510402. LA960918X